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"Electrophoresis and Immunoblotting". In: Current Protocols in Cell

1. Current Protocols in Cell Biology Electrophoresis and Immunoblotting 6 1 19 Supplement 37 One Dimensional SDS PAGE 6 1 20 Supplement 37 light solution heavy solution peristaltic pump e Tygon tubing gradient maker reservoir chamber mixing chamber interconecting valve stir bar vertical outlet valve gel unit pipet tip ring stand Figure 6 1 2 Gradient gel setup A peristaltic pump though not required will provide better control 3 Using the recipes in Table 6 1 10 prepare light and heavy acrylamide gel solutions without ammonium persulfate or TEMED Recommended gradient ranges are 5 to 20 for most applications to separate proteins of 10 to several hundred kilodaltons Deaeration is not recommended for either the light or heavy solution Omitting the deaeration will allow polymerization to proceed more slowly letting the gradient establish itself in the gel sandwich before polymerization takes place Keep the heavy solution on ice until use Once the ammonium persulfate is added to the heavy solution it will polymerize without TEMED albeit more slowly keeping the solution on ice prevents this The gel solution will come to room temperature during cast ing The higher the percentage of acrylamide the more severe the problem of premature polymerization With the outlet port and interconnecting valve between the two chambers closed pipet
2. Plikaytis B D Carlone G M Edmonds P and Mayer L W 1986 Robust estimation of stan dard curves for protein molecular weight and linear duplex DNA base pair number after gel electrophoresis Anal Biochem 152 346 364 Russ J C 1995 The Image Processing Handbook CRC Press Boca Raton Fla Smith J M and Thomas D J 1990 Quantitative analysis of one dimensional gel electrophoresis profiles Comput Appl Biosci 6 93 99 Sutherland J C Lin B Monteleone D C Mugavero J Sutherland B M and Trunk J 1987 Electronic imaging system for direct and rapid quantitation of fluorescence from electro phoretic gels Application to ethidium bromide stained DNA Anal Biochem 163 446 457 Welch T A 1984 A technique for high performance data compression IEEE Computer 17 21 32 KEY REFERENCES Glasbey and Horgan 1994 See above Describes general image processing techniques as they are applied to biological images Russ 1995 See above A general reference book on digital image capture and analysis Sutherland J C 1993 Electronic imaging of elec trophoretic gels and blots In Advances in Elec trophoresis Vol 6 A Chrambach M J Dunn and B J Radola eds pp 1 41 VCH Verlagsge sellschaft mbH Weinheim Germany Provides an overview of image capture with particu lar emphasis on types of capture equipment INTERNET RESOURCES rsb info nih gov nih image NIH Image is free softwa
3. 9 Clean pouring apparatus with water do not use detergent 10 Allow the gel to polymerize for at least 30 min at room temperature Remove the isobutyl alcohol wash twice with water and overlay with 1x BN gel buffer Store at 4 C stable at least 1 year Pour the stacking gel 11 Just before use pour the stacking gel using an appropriate comb and the BN stacking gel solution see Reagents and Solutions Due to the low percentage of acrylamide bisacrylamide the comb might be difficult to remove without damaging the gel Try to move the comb perpendicular to the plane of the glass plates while removing it to allow air to enter Alternatively the percentage of acrylamide bisacrylamide can be increased to 3 5 Load the samples and run the Blue Native gels All steps should be performed at 4 C e g in a cold room 12 Add 100x pervanadate solution to the dialysed lysate to a concentration of 1x if phosphorylation needs to be preserved 13 Mount the gel in the electrophoresis apparatus and load 5 to 30 ul dialysed cell lysate prepared as in Support Protocol in the dry wells of the mounted gel As control an aliquot of the sample can be boiled with 1 SDS to destroy all multiprotein complexes Leave one lane free between this control and the non SDS samples 14 Load 10 ul marker mix 1 and 10 ul marker mix 2 in two adjacent wells Only ferritin is seen during the electrophoresis due to its brown color Alternatively
4. The recipes produce 15 ml of separating gel and 5 ml of stacking gel which are adequate for a gel of dimensions 0 75 mm x 14 cm x 14cm The recipes are based on the SDS denaturing discontinuous buffer system of Laemmli 1970 Pall reagents and solutions used in the protocol must be prepared with Milli Q purified water or equivalent Volumes are in milliliters The desired percentage of acrylamide in the separating gel depends on the molecular size of the protein being separated See annotation to step 3 Basic Protocol 1 4Best to prepare fresh Failure to form a firm gel usually indicates a problem with the ammonium persulfate TEMED or both TEMED N N N N tetramethylethylenediamine Current Protocols in Cell Biology Table 6 1 2 Molecular Weights of Protein Standards for Polyacrylamide Gel Elect rophoresis Protein Molecular weight Da Cytochrome c 11 700 Lactalbumin 14 200 Lysozyme hen egg white 14 300 Myoglobin sperm whale 16 800 B Lactoglobulin 18 400 Trypsin inhibitor soybean 20 100 Trypsinogen PMSF treated 24 000 Carbonic anhydrase bovine erythrocytes 29 000 Glyceraldehyde 3 phosphate dehydrogenase rabbit muscle 36 000 Lactate dehydrogenase porcine heart 36 000 Aldolase 40 000 Ovalbumin 45 000 Catalase 57 000 Bovine serum albumin 66 000 Phosphorylase b rabbit muscle 97 400 8 Galactosidase 116 000 RNA polymerase E coli 160 000 Myosin heavy chain rabbit muscle 205 000 Protein
5. 1000 x leupeptin Prepare a 10 mg ml solution of leupeptin in water Store in 1 ml aliquots up to 5 years possibly longer at 20 C 1000 x aprotinin Prepare a 10 mg ml solution of aprotinin in water Store in 1 ml aliquots up to 5 years possibly longer at 20 C 100x PMSF Prepare a 100 mM solution of phenylmethylsulfonyl fluoride PMSF in ethanol Store in 1 ml aliquots up to 5 years possibly longer at 20 C 100x sodium orthovanadate Prepare a 50 mM solution of sodium orthovanadate in water Store up to 1 year possibly longer at room temperature 100x sodium fluoride Prepare 1 M solution of sodium fluoride in water Store up to 5 years possibly longer at room temperature COMMENTARY Background Information Most if not all proteins require binding to other proteins in order to fulfill their func tion Thus they form multiprotein complexes MPCs Most proteins are part of several dis tinct complexes as well as being present as monomers The abundance of distinct com plexes of which a certain protein is a subunit can vary enormously Furthermore complexes might have different stabilities and these can change over time and space Therefore iden tifying and analyzing complexes is a difficult task Common techniques to study complexes such as immuno precipitation UNIT 7 2 or two hybrid UNIT 17 3 methods allow the iden tification of binding partners of the protein of interest they howe
6. 9H 1 3 di chloro 9 9 dimethylacridin 2 one 7 yl phosphate diammonium salt to make 1 25 mg ml stock solution Store the stock solution at 20 C desiccated and protected from light When properly stored the stock solution should be stable for at least 6 months When the solution turns a blue color the substrate has broken down and is no longer usable Prepare the working solution fresh Fix solution Prepare a solution of 50 methanol and 50 deionized water Store up to 6 months at room temperature One 8 X 10 cm gel will require 100 ml of fix solution Incubation buffer Prepare blocking solution see recipe with 1 mM CaCl and 0 5 mM MgCl Store up to 6 months at room temperature Periodic acid solution Add 250 ml of 3 v v acetic acid to the bottle containing the periodic acid oxidizing solution and mix until completely dissolved Store up to 6 months at room temperature Sample buffer 2x 100 mM Tris Cl pH 6 8 APPENDIX 24 20 v v glycerol 4 w v sodium dodecy sulfate 0 1 w v bromophenol blue Store up to 6 months at room temperature Wash solution Prepare a solution of 3 v v glacial acetic acid in water Store up to 6 months at room temperature One 8 x 10 cm gel will require 400 ml of wash solution Wash solution IT Prepare a solution of 50 mM Tris Cl pH 7 5 150 mM NaCl Store up to 6 months at room temperature COMMENTARY Background Information The analysis
7. Contributed by Scott Medberry and Sean Gallagher Current Protocols in Cell Biology 2002 6 19 1 6 19 14 Copyright 2002 by John Wiley amp Sons Inc UNIT 6 9 Electrophoresis and Immunoblotting 6 9 1 Supplement 16 Digital Electrophoresis Analysis 6 9 2 Supplement 16 are performed in the same manner every time Allowing the computer to do repetitive tasks and complicated calculations minimizes the chance for individual errors This does not im ply that such measurements are correct just that they are reproducible An incorrect routine or algorithm can also invalidate data Cost A consideration when evaluating any labo ratory method is cost Digital electrophoresis analysis equipment can be expensive In many cases however it offers the only method for achieving acceptable analysis performance In other cases equal performance can be achieved using silver halide technology However tradi tional photography can also be expensive when the costs of consumable supplies such as film and developers as well as other expensive re quirements such as developing tanks and dark rooms are included Often digital methods can be a good choice when all costs are considered KEY TERMS FOR IMAGING There are several specialized terms encoun tered during digital image analysis The most commonly encountered are contrast bright ness gamma saturation resolution and dy namic range They describe con
8. Heating the proteins in the presence of SDS leads to their unfolding and to the disruption of multiprotein complexes 7 Fill the large pocket of the second dimension gel with 1 x SDS electrophoresis buffer removing all air bubbles Incline the gel with the large glass plate at the bottom Let the first dimension gel slice enter between the glass plates Avoid any air bubbles and push carefully with a spacer without added tape 8 Place the gel into the electrophoresis apparatus and load the markers and control samples into the small wells It is recommended to keep one third of the sample to be separated by the first dimension BN PAGE Support Protocol step 19 boil it in 1x SDS electrophoresis buffer and use it as a control sample in the second dimension 9 Overlay the first dimension gel strip with a 3 mm layer of 2x SDS sample buffer Run and analyze the second dimension SDS gel 10 Run and disassemble the gel as described in unr 6 1 Basic Protocol 1 The dye front contains not only the bromphenol blue of the SDS sample buffer but also the Coomassie blue from the first dimension The dye front of the first dimension will be visible as a dark blue spot at the bottom of the second dimension Current Protocols in Cell Biology Electrophoresis and Immunoblotting 6 10 9 Supplement 38 ALTERNATE PROTOCOL 1 Two Dimensional Blue Native Polyacrylamide Gel Electrophoresis 6 10 10 Supplement 38 11 Visual
9. resis are obtained A single person can conven iently run about 16 two dimensional gels in one week depending on the amount of electrophore sis equipment available The rate limiting step in most laboratories is running the second dimen sion gels because 16 soluble ampholyte IEF gels or 12 IPG gels can be focused in one run but loading running and detecting results from 12 to 16 second dimension gels requires substantial operator time and electrophoresis equipment Literature Cited Anderson N G and Anderson N L 1978a Two di mensional analysis of serum and tissue proteins Multiple isoelectric focusing Anal Biochem 85 331 340 Anderson N L and Anderson N G 1978b Two di mensional analysis of serum and tissue proteins Multiple gradient slab gel electrophoresis Anal Biochem 85 341 354 Celis J E Rasmussen H H Leffers H Madsen P Honore B Gesser B Dejgaard K and Vandekerckhove J 1991 Human cellular pro tein patterns and their link to gnome DNA se quence data Usefulness of two dimensional gel electrophoresis and microsequencing FASEB J 5 2200 2208 Dunbar B 1987 Troubleshooting and artifacts in two dimensional polyacrylamide gel electro phoresis Jn Two Dimensional Electrophoresis and Immunological Techniques B S Dunbar ed pp 173 195 Plenum New York Garrels J I 1979 Two dimensional gel electropho resis and computer analysis of proteins synthe sized by cloned cell
10. termined by comparing the mobility of the protein band to the mobility of the dye front see Support Protocol 4 CAUTION Before any protocols are used it is extremely important to read the following section about electricity and electrophoresis ELECTRICITY AND ELECTROPHORESIS Many researchers are poorly informed concerning the electrical parameters of running a gel It is important to note that the voltages and currents used during electrophoresis are dangerous and potentially lethal Thus safety should be an overriding concern A working knowledge of electricity is an asset in determining what conditions to use and in troubleshooting the electrophoretic separation if necessary For example an unusually high or low voltage for a given current milliampere might indicate an improperly made buffer or an electrical leak in the chamber Safety Considerations 1 Never remove or insert high voltage leads unless the power supply voltage is turned down to zero and the power supply is turned off Always grasp high voltage leads one at a time with one hand only Never insert or remove high voltage leads with both hands This can shunt potentially lethal electricity through the chest and heart should electrical contact be made between a hand and a bare wire On older or homemade instruments the banana plugs may not be shielded and can still be connected to the power supply at the same time they make contact with a hand With commercial modern po
11. 100 ml BSB Wash 1 hr with 200 to 300 ml BSB Immunodetect protein 25 Prepare a solution of dilute I labeled rabbit anti human vWF polyclonal antibody in BSB to a concentration of 2 x 10 cpm in a volume sufficient to cover the gel Add 0 5 ml of 2 human IgG to the antibody solution The majority of the gels can be covered with 50 to 70 ml In an effort to minimize the volume of radioactive solutions the authors laboratory keeps a supply of dedicated plasticware to accommodate a wide variety of gel sizes Strict laboratory precaution should be exercised in the preparation handling and disposal of radioactive materials 26 Incubate the gel in the antibody solution at least 16 to 24 hr with gentle mixing 27 Discard the antibody solution adhering to standard radiation safety waste disposal protocols 28 Wash the gel 1 hr in 200 ml high salt wash buffer with gentle mixing using a horizontal rotary mixer 29 Repeat the wash an additional three to four times The washes should be monitored and continued until all excess I is removed 30 Wash 1 hr in 200 ml distilled water Repeat once 31 Dry the gel with a forced hot air dryer directed on the gel or air dry the gel Autoradiograph the gel 32 Place the gel in a Kodak X Omatic film cassette with a Lanex screen Under dark room conditions place a piece of Kodak X Omat film on the gel and then incubate the cassette at 70 C Expose for an appropriate amount of t
12. 187 5 ul of 2 5 ammonium persulfate solution and swirl 5b Pipet the gel solution into the bottom of the glass cylinder Gently run water down the outside of the tube bundle using a wash bottle Keep adding water until the gel mix reaches the desired height Hydrostatic pressure will force the gel solution into the tubes Sufficient gel solution must be used to obtain the desired gel height while avoiding forcing any water into the tubes The volume of gel solution required can be estimated as follows number of gels X 3 14 x tube internal radius in cm x height in cm 10 ml to keep a safe level of gel mix at the bottom of the casting cylinder As water is less dense than the gel solution the water level will be slightly higher than the level of gel solution inside the tubes 6b Overlay the gels with 8 M urea Urea decomposes at a substantial rate at room temperature therefore the gels should be used the same day they are cast 7b Let the gels polymerize at least 3 hr prior to use Mount the gels in the electrophoresis unit 8 Prepare the lower electrode solution by degassing the proper amount of 0 1 M H PO under vacuum with stirring for at least 5 min Fill the bottom electrophoresis chamber The amount of phosphoric acid depends on the length of the gel tubes and the type of electrophoresis unit The solution should cover the entire gel for good heat dissipation Approximately 3 liters are required for Protean II xi 2D electrop
13. 1985 The specific interactions between labeled lectins and oligosaccharides form the basis of glycoprotein detection after separation by gel electrophoresis and transfer to membranes by electroblotting Concanavalin A is a tetrameric protein with each subunit containing a carbohydrate binding site a calcium ion binding site and a manganese ion binding site Concanavalin A binds specifically Current Protocols in Cell Biology BASIC PROTOCOL 2 Electrophoresis and Immunoblotting 6 8 7 Supplement 16 Fluorescence Detection of Glycoproteins in Gels and on Electroblots 6 8 8 Supplement 16 to O D mannopyranosyl and o D glucopyranosyl residues with substitutions or modifi cations at the C 3 C 4 or C 6 positions of the ring structure leading to greatly diminished binding Beeley 1985 The Pro Q Glycoprotein Blot Stain Kit with concanavalin A utilizes alkaline phosphatase conjugated concanavalin A along with the fluorogenic substrate DDAO phosphate 9H 1 3 dichloro 9 9 dimethylacridin 2 one 7 yl phosphate to detect glycoproteins on nitrocellulose and poly vinylidene difluoride PVDF membranes The detection proce dure is similar to that of standard western immuno blotting DDAO phosphate is rapidly converted to the long wavelength red fluorescent product DDAO DDAO absorbs maximally at either 275 nm or 646 nm and emits maximally at 659 nm Consequently the blots may be imaged using standard UV epi
14. A second dimension gel that is slightly thicker than the first dimension gel allows fitting in the first dimension gel slice without problems but still fixing it between the glass plates so that it cannot move 3 Pour the separating SDS PAGE gel as described in UNIT 6 1 For everyday purposes degassing of the gel solution is not necessary It is recommended to use multicasting equipment to pour several SDS gels at once It saves time and ensures best reproducibility for comparisons of multiple samples Casting of multiple gradient gels is described in UNIT 6 1 4 Pour the stacking gel UNIT 6 1 using combs that contain one large pocket that is wide enough to accommodate the first dimension gel slice and one or two additional wells for marker and control samples UNIT 6 4 After removing the comb clean the large pocket of polymerized gel remnants using a spacer that has not been enlarged with a layer of tape Load the Blue Native gel strips onto the second dimension SDS gels 5 Retrieve the first dimension BN gel strips from storage Incubate them for 10 min at room temperature in 2x SDS sample buffer by shaking in a small tray on a platform shaker Make sure to work under a hood if working with a reducing sample buffer containing 2 mercaptoethanol Current Protocols in Cell Biology BN PAGE BN PAGE SDS SS g o kDa 175 ADVd Sds J9Yd SAS top 880 440 bottom 880 ean top bottom Figure 6 10 1 T
15. PREPARING PROTEINS IN TISSUE SAMPLES Tissue samples are usually solubilized in lysis buffer using homogenization After centrifugation the protein sample can be loaded onto the first dimension gel In general much higher sample to sample variability is expected when tissue samples are analyzed Materials Tissue samples Lysis buffer see recipe Dounce homogenizer or equivalent Ultracentrifuge and rotor e g Beckman Ti70 2 C 1 Place tissue sample in a Dounce homogenizer add 2 ml lysis buffer per 100 mg tissue and homogenize the sample on ice e g 3 to 5 strokes 2 Let the mixture stand a few minutes then transfer to an appropriately sized ultracen trifuge tube depending on total sample volume 3 Centrifuge 2 hr at 100 000 x g e g 33 000 rpm in a Beckman Ti70 rotor for 100 000 x g or hr at 200 000 x g 2 C 4 Divide the supernatant into aliquots and freeze at 80 C or immediately load an appropriate volume onto the IEF gel SECOND DIMENSION ELECTROPHORESIS OF IEF TUBE GELS Second dimension gels are identical to those described in UNIT 6 1 except for sample loading which requires a broad flat well A broad well can be cast using an appropriate two dimensional comb if the second dimension gel thickness is slightly larger than that of the first dimension gel Alternatively when the second dimension gel is being cast water can be layered over the entire surface of the gel to produce a flat surface that wi
16. SDS PAGE UNIT 6 1 or affixed to filter membranes Autoradiography see Basic Protocol is the most common method by which this is accomplished and X ray film is the traditional recording medium The use of autoradiography with gels requires that the gel be dried prior to being placed in contact with the film see Support Protocol 1 The decay of radioactive materials within the dried gel or filter leaves an image on the film that reflects its distribution in the sample Film images can be quantified by densitometry see Support Protocol 4 to obtain a relative measure of the amount of radioactivity in the sample The use of X ray films for autoradiography however suffers from two drawbacks lack of sensitivity and a limited linear range over which the image density reflects the amount of radioactivity Lack of sensitivity can be overcome by fluorography see Alternate Protocol 1 or by the use of intensifying screens see Support Protocol 2 both of which enhance the radioactive signal Ensuring that the exposure is within a linear range requires careful controls film is often preflashed see Support Protocol 3 to increase the linear measurement range for weakly radioactive samples and it is important to ensure that the film not be saturated to attain strong radioactive signals Sensitivity and linear ranges of measurement can be greatly extended by using a phosphor imaging system see Alternate Protocol 2 Phosphor imaging also makes it much f
17. Suboptimal staining with SYPRO Ruby can be obtained by incubating the gel with stain solution for 60 to 90 min and skipping the subsequent washing steps Basic Protocol 3 steps 4 and 5 In the case of Coomassie blue staining binding of the dye to proteins is a fast process that depends mainly on its rate of diffusion into the gel matrix Hitchman and Ekstrom 1994 Removal of the unbound Coomassie dye from the gel which also relies on diffusion can be accelerated by placing an adsorbent material with affinity for the dye e g Whatman 3MM filter paper into the destaining solution with the gel or by in creasing the temperature of destaining e g to 37 C In both cases however caution should be taken to avoid excessive destaining that could decrease sensitivity Literature Cited Blum H Beier H and Gross H J 1987 Improved silver staining of plant proteins RNA and DNA in polyacrylamide gels Electrophoresis 8 93 99 Choi J K Na D S Hong H Y Choi D K Yoon S H and Yoo G S 1996 A fast and sensitive Coomassie blue staining for proteins in polyacrylamide gels using ion pairing agent Anal Lett 29 1517 1525 Cordoba O L Linskens S B Dacci E and San tom J A 1997 In gel cleavage with cyano gen bromide for protein internal sequencing J Biochem Biophys Methods 35 1 10 DeSilva T M 1995 Protein detection in gels using fixation In Current Protocols in Protein Science J E C
18. The appropriate user manuals should be consulted for specific details Since there is a greater selection of pH ranges for premade Immobiline DryPlates than DryStrips it is sometimes convenient to cut DryPlates into strips prior to rehydrating the gel to obtain narrower pH ranges where needed See Basic Protocol 4 for details concerning preparing and running the second dimension gel Wear gloves throughout the procedure and handle the Immobiline DryStrips with forceps where feasible to prevent extraneous protein contamination of the gels and gel solutions Current Protocols in Cell Biology BASIC PROTOCOL 2 Electrophoresis and Immunoblotting 6 4 9 Supplement 4 Two Dimensional Gel Electrophoresis 6 4 10 Supplement 4 Table 6 4 1 Rehydration Solutions for Immobiline DryStrips DryStrip type Component Final conc 3 10L 3 10NL 4 7L Ultrapure urea 7M 2 1g 21g 2 1g Thiourea 2M 0 76 g 0 76 g 0 76 g CHAPS 2 0 1 g 0 1 g 0 1 g Pharmalyte pH 3 10 1 50 100 ul Pharmalyte pH 4 6 5 50 ul 100 ul Pharmalyte pH 8 10 5 25 ul Ampholine pH 6 8 25 ul DTT 0 3 75 mg 75 mg 75 mg Bromphenol blue Trace A few grains A few grains A few grains Milli Q water To 5 ml To 5 ml To 5 ml Rehydration solutions should be prepared fresh immediately before use or stored as frozen aliquots and should be filtered using a 0 2 um filter Minimize total time the solution is at room temperature prior to use to minimi
19. This renders them capable of being reduced through the developing process to form silver metal a grain The silver grains on the film form the image The choice of film is critical for autoradiography Double coated films e g Kodak X Omat AR and Fuji RX contain two emulsion layers on either side of a polyester support and are most commonly used for autoradiography Laskey and Mills 1977 Double coated films are ideal for detecting the high energy B particles emitted by P and I Contributed by Daniel Voytas and Ning Ke Current Protocols in Cell Biology 1998 6 3 1 6 3 10 Copyright 1998 by John Wiley amp Sons Inc UNIT 6 3 BASIC PROTOCOL Electrophoresis and Immunoblotting 6 3 1 Supplement 10 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots 6 3 2 Supplement 10 since they can penetrate the polyester support and expose both emulsion layers These films are normally used with calcium tungstate CaWO intensifying screens at reduced temperature 70 C they are highly sensitive to the blue light emitted by these screens The green light sensitive BioMax MS film Kodak is a double coated film spectrally matched to the blue and green light emitting BioMax MS intensifying screen The BioMax MS film BioMax MS intensifying screen system normally gives greatest sensi tivity to P four times greater than that of X Omat AR film with CaWO screens Single coate
20. i e after immuno precipitation The amount of Triton X 100 in the lysis buffer is normally sufficient for effective dissociation of SDS from proteins If poor results are encountered with SDS containing samples try decreasing the final SDS concentration and or increasing the final Triton X 100 concentration in the sample If no proteins are detected on the gel check whether 1 the total protein load is appropriate for the detection method used 2 the orientation of electrical connections is wrong or electrical connection during isoelectric focusing is poor all gels from that run will be blank 3 an air bubble obstructs current in a single IEF tube or 4 the electrical connection is incorrect or is poor during the second dimension gel separation Careful monitoring of current and voltage at the begin ning during and at the end of electrophoretic separations is strongly recommended Recording the initial and final current and voltage will also facilitate troubleshooting Additional guidelines for troubleshooting and evaluating artifacts in two dimensional gel electrophoresis are de scribed by Dunbar 1987 Anticipated Results A two dimensional electrophoretic separation of proteins should produce a pattern of round or elliptical spots separated from one another The pI range of the separated proteins as well as the observed molecular weight range depend on the first dimension isoelectric focusing protocol and the percent
21. in gel procedure for the electrophoresis of very high molecular weight proteins and provides examples of its useful ness in understanding the structure of von Wille brand factor Shainoff 1993 See above This reference provides general background and the rationale for the use of modified ararose for the separation and identification of large proteins by electrophoresis Contributed by Dennis M Krizek and Margaret E Rick National Institutes of Health Bethesda Maryland Electrophoresis and Immunoblotting 6 7 13 Supplement 15 Fluorescence Detection of Glycoproteins in Gels and on Electroblots The co translational and post translational covalent attachment of oligosaccharides to proteins is acommon cellular event in eukaryotes regulated by a variety of glycosidases and glycosyltransferases Beeley 1985 Reuter and Gabius 1999 unit 15 2 Glycosyla tion profiles are dynamic changing during development differentiation and disease Glycosylation of proteins is critical to the adhesiveness of microorganisms and cells cellular growth control cell migration tissue differentiation and inflammatory reactions Differences in glycosylation profiles are often used as a barometer to assess disease states With the advent of proteomics genome wide protein analysis there is renewed interest in the rapid and sensitive identification of glycoproteins by methods that do not require degradation of the protein com
22. is incorporated Citations and annotations to this are retrieved as well All of this information is compiled automatically into an interactive report about the protein From this report the researcher can formulate a more refined hypothesis and plan the most appropriate experiments to test it LITERATURE CITED Appel R D Hochstrasser D F Funk M Vargas J R Muller A F and Scherrer J R 1991 The MELANIE project From a biopsy to automatic protein map interpretation by computer Electro phoresis 12 722 735 Garrels J I 1989 The QUEST system for quantita tive analysis of two dimensional gels J Biol Chem 264 5269 5282 Glasbey C A and Horgan G W 1994 Image Analysis for the Biological Sciences John Wiley amp Sons Chichester England Hamming R W 1973 Numerical methods for sci entists and engineers 2nd ed Dover Publica tions New York Huffman D A 1952 A method for the construction of minimum redundancy codes Proc Inst Elect Radio Eng 40 9 12 Monardo P J Boutell T Garrels J I and Latter G I 1994 A distributed system for two dimen Electrophoresis and Immunoblotting 6 9 13 Supplement 16 Digital Electrophoresis Analysis 6 9 14 Supplement 16 sional gel analysis Comput Appl Biosci 10 137 143 Patton W F 1995 Biologist s perspective on ana lytical imaging systems as applied to protein gel electrophoresis J Chromatogr A 698 55 87
23. otherwise there will be a gradual diffusion driven mixing of buffers between the two gels which will cause a loss of resolution The protein of interest should be present in 0 2 to 1 ug amounts in a complex mixture of proteins if the gel will be stained by Coomassie blue UNIT 6 6 Typically 30 to 50 ug of a complex protein mixture in a total volume of lt 20 ul is loaded on a 0 75 mm thick slab gel 16 cm 10 wells When casting multiple gradient gels elim inate all bubbles in the outlet tubing of the gradient maker If air bubbles get into the out let tube they may flow into the caster and then up through the gradient being poured causing an area of distortion in the polymerized gel Air bubbles are not so great a problem when casting single gradient gels from the top As the gels are cast the stirrer must be slowed so that the vortex in the mixing chamber does not allow air to enter the outlet Uneven heating of the gel causes differ ential migration of proteins with the outer lanes moving more slowly than the center lanes called smiling Increased heat transfer elim inates smiling and can be achieved by filling the lower buffer chamber with buffer all the way to the level of the sample wells by main taining a constant temperature between 10 to 20 C and by stirring the lower buffer with a magnetic stirrer Alternatively decrease the heat load by running at a lower current If the tracking dye band is diffuse p
24. see Basic Protocol 1 and analysis of Blue Native gels by immunoblotting Alternate Protocol 1 or 2 Prepare the samples and load the Blue Native gel 1 Prepare a dilution series of the monoclonal antibodies of interest using the BN dialysis buffer containing the same detergent as the sample Start with 1 ug of antibody in 2 ul in the first reaction tube then dilute 1 10 1 100 1 1 000 and 1 10 000 in another four tubes Place the tubes on ice Antibodies are mostly dissolved in PBS and thus the nondiluted antibody will be present with sodium chloride If only 2 ul are used the resulting salt concentration is tolerated by BN PAGE If however more than 2 ul have to be used it is recommended to dialyze the antibody against BN dialysis buffer without any detergent Support Protocol Use fresh antibodies that have not been frozen and thawed too often in order to avoid the presence of antibody aggregates If possible use Fab fragments of the antibodies 2 Use any sample as described in the Support Protocol e g a dialysed lysate or purified proteins Add 10 to 20 ul of sample to each antibody containing tube mix and incubate 20 min on ice 3 Cast and pour the BN gel as in Basic Protocol 1 Use an acrylamide concentration Table 6 10 1 in which the multiprotein complex of interest is located on the lower third of the gel after the electrophoresis The reason for this is that all antibody shifts will be above the protein co
25. 0 054 Acrylamide concentration in heavy gel solution Stock solution 10 11 12 13 14 15 16 17 18 19 20 30 acrylamide 0 8 55 6l 66 72 77 83 88 94 99 105 110 bisacrylamide 4x Tris Cl SDS pH 41 41 41 41 41 41 41 A 41 41 41 8 8 H20 Sucrose g persulfate TEMED 10 ammonium 55 25 50 25 44 25 39 25 33 25 28 25 22 25 17 11 55 0 25 25 25 25 0 55 0 55 0 55 0 55 0 55 0 55 0 55 0 55 0 55 0 55 0 55 0 054 0 054 0 054 0 054 0 054 0 054 0 054 0 054 0 054 0 054 0 054 To survey proteins gt 10 kDa 5 to 20 gradient gels are recommended To expand the range between 10 and 200 kDa a 10 to 20 gradient gel is recommended Volumes are in milliliters sucrose is in grams Recipes produce ten 1 5 mm thick gradient gels with 10 ml extra solution to account for losses in tubing Keep light gel solution at room temperature prior to use no longer than 1 hr Keep heavy solution on ice See Table 6 1 1 for preparation 4Best to prepare fresh Failure to form a firm gel usually indicates a problem with the ammonium persulfate TEMED or both Current Protocols in Cell Biology gradient maker reservoir chamber heavy solution mixing chamber light solution outlet valve mixing valve outlet port inlet port stir bar stir plate peristaltic pump multiple gel caster Figure 6 1 3 Setup for casting multiple gradient gels Casting multipl
26. 5 30 35 40 45 50 55 60 65 70 bisacrylamide 4x Tris Cl SDS 3 75 3 75 3 75 3 75 3 75 3 75 3 75 375 3 75 3 75 pH 8 8 H20 8 75 8 25 7 75 7 25 6 75 6 25 5 75 5 25 4 75 4 25 10 ammonium 0 05 0 05 0 05 0 05 0 05 0 05 0 05 0 05 0 05 0 05 persulfate TEMED 0 005 0 005 0 005 0 005 0 005 0 005 0 005 0 005 0 005 0 005 Acrylamide concentration in heavy gel solution b Stock solution 10 11 12 13 14 15 16 17 18 19 20 30 acrylamide 0 8 5 0 55 60 65 70 75 80 85 90 95 10 0 bisacrylamide 4x Tris Cl SDS 3 75 3 75 3 75 3 75 3 75 3 75 BII 3 75 3 75 3 75 3 75 pH 8 8 H20 50 45 40 35 30 25 20 15 10 0 5 0 Sucrose g 2 25 2 25 2 25 2 25 2 25 2 25 2 25 2 25 2 25 2 25 2 25 10 ammonium 0 05 0 05 0 05 0 05 0 05 0 05 0 05 0 05 0 05 0 05 0 05 persulfate TEMED 0 005 0 005 0 005 0 005 0 005 0 005 0 005 0 005 0 005 0 005 0 005 To survey proteins gt 10 kDa 5 to 20 gradient gels are recommended To expand the range between 10 and 200 kDa a 10 to 20 gradient gel is recommended Volumes are in milliliters sucrose is in grams Keep light gel solution at room temperature prior to use no longer than 1 hr Keep heavy solution on ice See Table 6 1 1 for preparation d Ammonium persulfate and TEMED are added directly to the gradient chambers immediately before the gel is poured It is best to prepare ammonium persulfate fresh Failure to form a firm gel usually indicates a problem with the ammonium persulfate TEMED or both
27. 6 10 1 Add APS and TEMED only immediately before use The volumes of the two solutions combined should be exactly equal to the volume required to fill the glass plate sandwich to the required height When using multicasting equipment the dead volume of the apparatus has to be deter mined empirically and added to the required volume of the gel solutions 5 Pour the low percentage light BN separating gel solution into the reservoir cham bers and the high percentage heavy BN separating gel solution into the mixing chamber of the gradient mixer Fig 6 1 2 6 Open the interconnecting valve and force out the air bubble inside the connecting channel by pressing over the right cylinder with your thumb CAUTION Make sure to wear gloves in order to avoid contact with acrylamide Cast the Blue Native separating gels 7 Switch on the peristaltic pump open the outlet valve and allow the gel to slowly enter between the glass plates Ensure that the needle at the end of the Tygon tubing is always above the height of the liquid so that the gradient is not disturbed When using multicasting equipment the gel solutions enter between the glass plates from the bottom In this case the low percentage BN separating gel solution has to be placed into the mixing chamber and the high percentage one into the reservoir chamber Fig 6 1 3 Figure 6 1 2 shows a pipet tip at the end of the Tygon tubing but a needle may be more appropriate since a p
28. 6 6 1 shows a comparative experi ment in which serial dilutions of protein stand ards were run on SDS PAGE unir 6 1 and subsequently stained following the four Basic Protocols described in this unit Among the four methods silver Fig 6 6 1B and Coomassie blue staining Fig 6 6 1A gave the highest and lowest sensitivity respectively The detection limit of fluorescent staining with SYPRO Ruby could be greatly enhanced by the integration effect of a CCD or photographic camera Thus while by direct observation on a UV transillu mination unit SYPRO Ruby staining was sig nificantly less sensitive than negative zinc staining both methods gave comparable sensi tivities upon the use of a CCD camera to inte grate the signal of SYPRO Ruby stained bands Fig 6 6 1C and D Simplicity While staining with Coomassie blue SYPRO Ruby or zinc are relatively simple methods silver staining involves a number of steps that need to be carefully controlled most notably sensitization with thiosulfate Basic Protocol 2 step 6 rinsing with water before and after silver impregnation Basic Protocol 2 steps 7 to 8 and 10 to 11 respectively and image development Basic Protocol 2 step 13 In fact the duration of the image development step critically influences the sensitivity of silver staining thus resulting in experiment to ex periment variations in band color intensity Staining with SYPRO Ruby requires a UV transilluminator to vi
29. 6 months Prepare the cell lysates 8 9 10 Resuspend the cell pellet in ice cold BN lysis buffer or other lysis buffer that has been used successfully to extract your multiprotein complex of interest The concentration of cells per volume lysis buffer depends on the amount of protein that should be loaded on the BN gel As an approximation use 10 cells per 250 to 500 ul lysis buffer Incubate on ice for 15 min to allow cell lysis The lysis time can be prolonged if it is convenient e g in the case where many different samples are processed and some of them have to wait for the last ones Microcentrifuge 15 min at 13 000 x g 4 C to remove insoluble material Prepare to dialyze the lysate 11 12 13 14 15 16 17 Melt a hole in the cap of a microcentrifuge tube using the large diameter end of a hot Pasteur pipet Chill the tube on ice Transfer the supernatant from step 10 into the chilled tube from step 11 Place a dialysis membrane over the opened tube and close the cap Make sure that there are no folds or tears in the dialysis membrane Seal the cap at the edges with Parafilm Make sure that the hole in the tube is not covered by the Parafilm Invert the tube and centrifuge upside down at the lowest speed possible in the adaptor cavity for 50 ml tubes in a cell culture centrifuge for 10 sec at 4 C Prepare a 100 ml beaker with ice cold BN dialysis buffer and a magnetic stirrer Us
30. ESA s ampholytes pH 3 10 2D are suited for most applications and give reproducible results Although purity of all reagents is important the purity of urea and choice of ampholytes are among the most critical factors for the quality and performance of isoelectric focusing Most commercially available reagents marketed specifically for two dimensional gel electrophoresis should be suitable although individual lots of reagents from any supplier may provide variability and or unacceptable results Cast gels by pouring 3a Wrap one end of each glass tube with Parafilm and mount the tube in a casting stand Mark all the tubes to indicate the desired gel height For reproducible results all gels should be the same height 4a Filter the gel solution using a 10 ml syringe equipped with a syringe tip filter capsule Briefly degas the gel solution 5 min either by sonication or under vacuum Then add 42 5 ul TEMED and 187 5 ul of 2 5 w v ammonium persulfate solution to the filtered gel mixture and swirl gently to mix Current Protocols in Cell Biology Electrophoresis and Immunoblotting 6 4 3 Supplement 4 Two Dimensional Gel Electrophoresis 6 4 4 Supplement 4 5a Using a 10 ml syringe with a blunt needle fill each glass tube with gel solution to the desired height Make sure there are no air bubbles trapped in the gel A needle is the best choice for casting gels if tubes of 3 mm inner diameter are used Fo
31. Flattened no 2 cork borer Forceps fine Horizontal rotary mixer Forced hot air dryer optional Kodak X Omatic film cassette with Lanex screens and film CAUTION When working with radioactivity take apptopriate precautions to avoid contamination of the experimenter and the surroundings Carry out the experiment and dispose of wastes in an appropriately designated area following the guidelines provided by your local radiations safety officer also see APPENDIX 1D Prepare sample 1 2 Prepare 3 ml fresh in gel sample buffer Label a 12 x 75 mm polypropylene tube for each sample to be assayed Add 20 ul borate saline buffer BSB 120 ul sample buffer and 10 ul sample into each tube Cover each tube vortex gently and incubate 2 hr at 37 C 4 Add 8 ul of 0 5 bromphenol blue to each sample and mix gently Prepare agarose gel 5 10 11 Prepare a boiling water bath by placing approximately 50 ml distilled water in a 250 ml beaker and incubating on a hot plate with magnetic stirring capabilities Use isopropanol to clean the 12 5 x 26 0 x 0 3 cm glass plate and 12 5 x 24 0 cm spacer plate with its adherent 0 5 mm spacers Dry with a lint free tissue e g Kimwipe It is advisable because of the fragile nature of this gel to prepare sufficient materials to pour a gel in reserve in the eventuality that one is rendered unusable Place a few milliliters of distilled water on the glass plate and adh
32. J R and Osborn M 1972 Measurement of molecular weights by elec trophoresis on SDS acrylamide gel Methods Enzymol 26 3 27 Key Reference Hames B D and Rickwood D eds 1990 Gel Electrophoresis of Proteins A Practical Approach 2nd ed Oxford University Press New York An excellent book describing gel electrophoresis of proteins Current Protocols in Cell Biology Immunoblotting and Immunodetection Immunoblotting often referred to as western blotting is used to identify specific antigens recognized by polyclonal or monoclonal antibodies Protein samples are solubilized usually with sodium dodecyl sulfate SDS and reducing agents such as dithiothreitol DTT or 2 mercaptoethanol 2 ME Following solubilization the material is separated by SDS PAGE unir 6 1 The antigens are then electrophoretically transferred in a tank see Basic Protocol 1 or a semidry transfer apparatus see Alternate Protocol 1 to a nitrocellulose PVDF or nylon membrane a process that can be monitored by reversible staining see Support Protocol 1 or by Ponceau S staining see Support Protocol 2 Previously stained gels may also be blotted see Alternate Protocol 2 The transferred proteins are bound to the surface of the membrane providing access for reaction with immunodetection reagents All remaining binding sites are blocked by immersing the membrane in a solution containing either a protein or detergent blocking agent After probing
33. Milli Q water Blot with a Kimwipe or tissue paper to remove excess water The electrode strips should be evenly soaked and just damp after blotting Excessive water could cause sample streaking Transfer the strips from step 6 to adjacent grooves in the aligner tray Position the rounded acidic end of each strip near the top of the tray at the red electrode anode near the cooling tubes and the square end at the bottom of the tray near the black electrode cathode Be sure that the edges of all gel strips at the anode end are lined up evenly Place the blotted electrode strips from step 9 on top of the gel surface of the DryStrips near the anode and cathode ends of the gel Position the red anode and black cathode electrodes on top of the electrode strips at their respective ends After the electrodes have been pressed down on top of the electrode strips check that the gel strips have not shifted position Push the sample cups onto the sample cup bar Place the sample cup bar near the anode end of the gel so that the small spacer arm just touches the electrode and the sample cups are nearest to the electrode but do not allow the cups to touch the gel The sample cups should face the nearest electrode The acidic end of the gel can usually be used for sample application however the optimal loading position may need to be determined empirically for different types of samples At high protein concentrations and or at non
34. Protocols in Cell Biology system the apparent pI of a given protein may be slightly different from that determined by other methods Therefore it is recommended that a broad range gradient be tried initially followed by a narrower range gradient if needed Diagonal gel electrophoresis Diagonal gel electrophoresis is a form of two dimensional analysis useful for investigating the subunit composition of multisubunit proteins containing interchain disulfide bonds Goverman and Lewis 1991 Proteins are electrophoresed in the first dimension in a tube gel or a slab gel under nonreducing conditions The proteins are then reduced in situ and the first gel or a strip thereof is layered onto a second gel and electro phoresed In the second gel the proteins migrate at right angles to the original first dimension migration Most cellular proteins are not disul fide linked and will fall on the diagonal in this system that is they migrate approximately equal distances in both directions during electrophore sis and lie approximately on the diagonal line connecting opposite comers of the gel On reduc tion component subunits of proteins connected by interchain disulfide bonds will resolve below the diagonal because the individual subunits mi grate faster than the disulfide linked complex during the second electrophoresis Some proteins with internal disulfide bonds but no interchain disulfides may migrate slightly above the
35. Rickwood eds pp 1 147 Ox ford University Press New York Current Protocols in Cell Biology Hedrick J L and Smith A J 1968 Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis Arch Biochem Biophys 126 155 164 Rodbard D and Chrambach A 1971 Estimation of molecular radius free mobility and valence using polyacrylamide gel electrophoresis Anal Biochem 40 95 134 Schagger H 1994 Native gel electrophoresis Jn A Practical Guide to Membrane Protein Purifica tion G Von Jagow and H Schagger eds pp 81 104 Academic Press San Diego Sigma 1986 Nondenatured protein molecular weight marker kit Technical Bulletin No MKR 137 Sigma Chemical Company St Louis Mo Key Reference Andrews 1986 See above Covers a variety of electrophoretic techniques in cluding nondenaturing electrophoresis and Fer guson plots Contributed by Sean R Gallagher Motorola Corporation BioChip Systems Tempe Arizona Electrophoresis and Immunoblotting 6 5 11 Supplement 5 Staining Proteins in Gels Polyacrylamide gel electrophoresis PAGE is a powerful method for the resolution of protein mixtures according to the proteins charge to mass ratio nondenaturing PAGE UNIT 6 5 mass SDS PAGE UNIT 6 1 isoelectric point isoelectric focusing or combina tions of these properties two dimensional PAGE unir 6 4 A number of protocols have
36. TEMED or both Current Protocols in Cell Biology Electrophoresis and Immunoblotting ee 6 1 7 Supplement 37 Table 6 1 3 Protein Standard Mixtures Available from Selected Suppliers Applications 1 D 2 1 Im Pref Fluor Gly Phos Bio Tag IEF Nat Bio Rad CalBiochem Cell Signaling Technology Favorgen GE Healthcare Invitrogen NEB Norgen Biotek Novagen PerkinElmer Pierce Promega Qiagen R amp D Systems Roche Applied Science Sigma Aldrich Upstate USB X X gt lt ee e a e KK KM OM X X X X X X X X gt lt gt lt x Kx KK gt lt X X X X X X X X X X X X X Abbreviations 1 D one dimensional gels 2 D two dimensional gels Im immunoblotting Pre prestained Fluor fluorescent Gly glycoprotein Phos phosphoprotein Bio biotinylated Tag tagged IEF isoelectic focusing Nat native b2 D standards are useful as independently characterized internal controls or reference standards for 2 D SDS PAGE Many investigators simply use an internally characterized test sample as a reference set Prestained standards while not as sharply delineated as unstained standards can be used to monitor progress of the separation since the bands are visible through the gel cassette during electrophoresis They are also useful for marking the position of a band after electroblotting to a nitrocellulose or PVDF membrane prior to immunoassay or ana
37. Turn off power supply and remove the electrodes once the dye front has reached the bottom of the gel Extrude and store Blue Native gels 19 20 21 22 Remove the glass plates with the gel in between from the electrophoresis apparatus Open the glass plates by lifting the smaller one and keeping the gel attached to the bigger bottom plate Remove the stacking gel with the smaller glass plate Cut away the lanes where the marker mixes were loaded from the rest of the gel using the smaller glass plate Visualize the marker proteins by standard Coomassie blue or silver staining UNIT 6 6 For some purposes the ferritin marker 440 and 880 kDa which is visible without Staining is sufficient When assigning the positions of the marker take into consideration that the gel might shrink upon staining Alternatively after the first dimension BN PAGE proteins can be stained directly with silver or Coomassie brilliant blue UNIT 6 6 or detected by immunoblotting western blotting Alternate Protocols I and 2 Optionally stained spots can be cut out and the proteins identified by mass spectroscopy Stamp out each individual lane with the smaller glass plate Either immediately run the second dimension SDS PAGE Basic Protocol 2 or freeze each lane wrapped in aluminum foil at 20 C stable at least 1 year Do not bend a frozen gel piece since it breaks easily Alternatively the first dimension BN gel can be transferre
38. acry lamide can help minimize potential reagent as sociated problems In most cases the best stand ard for a given two dimensional gel system is an experimental sample or control that is available in sufficient quantity so that many replicate aliquots can be frozen and stored for an extended time at 80 C alternatively a sample that can be reproducibly prepared over a long time frame would make an acceptable standard Such an experimental standard or reference is more likely to detect subtle but experimentally important changes in the two dimensional gel system than commercially available IEF or SDS gel standard mixes Another critical factor is equilibration of the first dimension gel in the second dimension equilibration buffer During this step urea dif fuses out of the IEF gel while SDS and reducing reagent diffuse into the gel If the gel is inade quately saturated with SDS vertical streaking will result However if the gel is incubated in the equilibration buffer for an extended time a sub stantial amount of the protein can rapidly diffuse out of the large pore IEF gel Losses arising from diffusion can be critical for any experiment be cause different proteins will diffuse at different rates but rigorous control of the incubation step is especially important if quantitative compari sons among gels are planned The simplest method of controlling the incubation time is to freeze the extruded IEF gel after a carefully
39. appropriate filters Additional reagents and equipment for SDS polyacrylamide gel electrophoresis UNIT 6 1 Current Protocols in Cell Biology Run gel 1 Prepare the protein samples of interest e g crude protein isolates cell lysates serum partially purified plasma membranes for SDS polyacrylamide gel electrophoresis Typically the protein sample is diluted to 10 to 100 ug ml with 2x sample buffer heated for 4 to 5 min to 95 C and 5 to 10 ul of diluted sample is then applied per gel lane for x 10 cm gels Larger gels require proportionally more material For convenience CandyCane glycoprotein molecular weight standards may also be applied to a lane or two Typically 2 ul of this standard is diluted in 6 ul of sample buffer and heated in the same manner as the samples to be characterized These standards contain a mixture of glycosylated and non glycosylated proteins ranging from 14 to 180 kDa in molecular weight The standards serve as molecular weight markers and provide alternat ing bands as positive and negative controls for glycoprotein and total protein detection Each protein is present at 0 5 mg ml 2 Separate proteins by SDS polyacrylamide gel electrophoresis using standard meth ods UNIT 6 1 The staining procedure is optimized for gels that are 0 5 to 1 mm thick Fix gel 3 After electrophoresis fix the gel by immersing it in 75 to 100 ml of fix solution in a polystyrene staining dish and incubating with
40. be recom mended for all experiments Characterization Once lanes and bands have been detected it is possible to interpret the mobility of the nu cleic acid or protein bands Depending on the method of electrophoretic separation informa tion on mass length or size pI or relative mobility R can be inferred from mobility information The mobility is characterized us ing a standard curve with internal standards of known properties The type of curve depends on several parameters By definition with Rr based separation a linear first order curve is used since it represents the linear relationship between mobility and Ry Similarly pI and mobility are generally linear in isoelectric fo cusing separations For separations based on size a curve generated from mobility versus the log of the molecular weight provides a relatively good fit as measured by the correla tion coefficient R2 Several other curves have been suggested for size based separations in cluding modified hyperbolic curves and curves of mobility versus molecular weight gt that have good correlation coefficients Plikaytis et al 1986 In some cases no single curve equa tion can adequately represent the data and methods of fitting smooth contiguous curves using only neighboring points such as a La grange or spline fit described in Hamming 1973 are necessary This is most common for size separations with a very large range of separation sizes and w
41. been developed to visualize the separated protein bands following electrophoresis In this unit four different methods which are widely used in cell biology laboratories are described These are staining with the dye Coomassie blue Basic Protocol 1 and Alternate Protocol 1 silver staining Basic Protocol 2 fluorescent staining with SYPRO Ruby Basic Protocol 3 and negative staining with zinc Basic Protocol 4 Staining with Coomassie blue Basic Protocol 1 is a relatively simple and very popular method albeit less sensitive than other procedures A variation of this protocol suitable for staining proteins in isoelectric focusing gels is described under Alternate Protocol 1 Staining with silver Basic Protocol 2 is probably the most sensitive method although it is time consuming and incompatible with some downstream applications see Com mentary The remaining two methods Basic Protocols 3 and 4 are more sensitive that Coomassie blue staining and recommended for downstream applications where protein fixation needs to be avoided e g analysis of biological activity Some of the protocols can be combined For instance some researchers prefer to stain gels first with Coomassie blue Basic Protocol 1 or zinc Basic Protocol 4 and if higher sensitivity is necessary subsequently with silver Basic Protocol 2 Further details on the differences between the methods as well as guidelines for their selection according to specific appli
42. can be visualized using a 300 nm UV B transilluminator 11 Document results before proceeding to the next step Use a photographic camera or CCD camera and the appropriate filters to obtain the greatest sensitivity see Fig 6 8 1 Pro Q Emerald dye signal will fade after SYPRO Ruby dye staining Current Protocols in Cell Biology Electrophoresis and Immunoblotting 6 8 3 Supplement 16 Fluorescence Detection of Glycoproteins in Gels and on Electroblots 6 8 4 Supplement 16 82 0 66 0 66 0 45 0 p T 2 310 31 0 2 a o 7 21 5 2 18 0 x O 140 14 0 6 5 82 66 0 888 45 0 a S 31 0 31 0 E D o 21 5 18 0 oO 2 Oo 14 0 14 0 6 5 Figure 6 8 1 Sensitivity and specificity of glycoprotein detection in 13 SDS polyacrylamide gels using Pro Q Emerald 300 glycoprotein gel stain A Detection of glycoproteins using Pro Q Emerald 300 glycoprotein gel stain B Detection of the total protein profile using SYPRO Ruby protein gel stain Lanes 1 and 2 broad range molecular weight markers containing the 45 kDa glycoprotein ovalbumin 1000 and 250 ng respectively Lane 3 blank Lanes 4 to 12 CandyCane molecular weight markers a mixture of glycosylated and unglycosylated proteins 1000 to 3 9 ng as two fold serial dilutions Gels were imaged using a Lumi Imager F1 instrument Roche Mol
43. complexes is a cellular lysate or tissue extract Preparation of a cellular lysate suitable for separation by BN gels is done by dialysis to remove any cations and metabolites see Support Protocol A detailed protocol for pouring and running of the BN gradient gels see Basic Protocol 1 and denaturation of the separated proteins followed by separation using a second dimension SDS PAGE see Basic Protocol 2 is given Visualization of the resulting two dimensional gel can be done according to standard protocols including general protein stains UNIT 6 6 and immunoblotting often referred to as western blotting UNIT 6 2 A second dimension is not always required Proteins can be detected after BN PAGE by such standard procedures as general protein stains or transfer of the proteins to a Current Protocols in Cell Biology 6 10 1 6 10 21 March 2008 Published online March 2008 in Wiley Interscience www interscience wiley com DOI 10 1002 047 1 143030 cb0610s38 Copyright 2008 John Wiley amp Sons Inc UNIT 6 10 Electrophoresis and Immunoblotting es 6 10 1 Supplement 38 BASIC PROTOCOL 1 Two Dimensional Blue Native Polyacrylamide Gel Electrophoresis 6 10 2 Supplement 38 membrane under denaturing see Alternate Protocol 1 or native see Alternate Protocol 2 conditions followed by immunodetection Using these protocols there is a special requirement for the antibodies used to detect the proteins of inte
44. con trolled 5 min incubation in the equilibration buff er any additional equilibration incubation time required can then be incorporated and carefully controlled when the sample is thawed for loading onto the second dimension gel Another crucial step is loading of the equili brated IEF gel onto the top of the second dimen sion gel Any irregularity or obstruction between the two gels including particles of dirt or air bubbles will affect the flow of current and disrupt Current Protocols in Cell Biology the resolution of proteins in the final gel Simi larly poor contact or any movement of the IEF gel during electrophoresis of the proteins out of the IEF gel into the second dimension gel will lead to artifacts Therefore it is advisable to embed the IEF gel in a buffered agarose matrix to ensure good electrical contact be tween the gels and to prevent gel movement after electrophoresis is initiated Finally the choice of second dimension gel composition and separation conditions can influ ence the quality of results A proper percentage of acrylamide should be selected to optimize resolution within the desired molecular mass range If gradient gels are needed use of a multi ple gel casting stand is the best way to ensure reproducibility among samples within a single experiment Further details on optimization of two dimensional gel systems are presented by Hochstrasser et al 1988 When no technical sample related or
45. determined empirically although the most universal condi tions are those described in the protocols in this unit In contrast IEF systems that do not use any detergents or denaturants are limited to that fairly small percentage of proteins which maintain good solubility near their isoelectric point Current Protocols in Cell Biology If high molecular weight proteins are ex pected but are not present in the final two dimen sional gel check the sample preparation protocol as well as the sample storage conditions The most likely problem is proteolysis during sample preparation Multiple freeze thawing cycles could contribute to this problem Vertical smears on the two dimensional gel suggest 1 insuffi cient equilibration of the IEF gel notenough SDS bound to the proteins 2 poor contact between the IEF and second dimension gels or 3 prob lems related to the stacking gel too short or wrong buffer Use of a stacking gel is especially impor tant when large diameter IEF gels are loaded onto smaller second dimension gels Omission of Triton X 100 or other nonionic or zwitterionic detergent from the final sample loaded on the gel can yield poor results especially for samples containing SDS because the amount of detergent in the IEF gels alone may be insuf ficient to remove bound SDS from proteins The presence of Triton X 100 in the sample is espe cially important when SDS sample buffer is used to solubilize protein samples
46. di mensional gels UNIT 6 1 Because transfer efficiency depends on many factors e g gel concentration and thickness protein size shape and net charge results may vary Below is a guideline for 0 75 mm thick SDS PAGE gels transferred by semidry blotting Percent acrylamide Size range transferred resolving gel 100 efficiency 5 7 29 150 kDa 8 10 14 66 kDa A 13 15 lt 36 kDa Immunoblotting and 18 20 lt 20 kDa Immunodetection 6 2 4 Current Protocols in Cell Biology buffer soaked filter paper gel membrane transfer buffer soaked filter paper stack cellophane gel membrane transfer buffer soaked filter paper stack buffer soaked filter paper Mylar mask anode Figure 6 2 2 Immunoblotting with a semidry transfer unit Generally the lower electrode is the anode and one gel is transferred at a time A Mylar mask optional in some units is put in place on the anode This is followed by three sheets of transfer buffer soaked filter paper the membrane the gel and finally three more sheets of buffer soaked filter paper To transfer multiple gels construct transfer stacks as illustrated and separate each with a sheet of porous cellophane For transfer of negatively charged protein the membrane is positioned on the anode side of the gel For transfer of positively charged protein the membrane is placed on the cathode side of the gel Transfer is achieved by applying a
47. each sandwich in the caster with stacking gel solution 10 Insert a comb into each sandwich and let the gel polymerize for 2 hr Insert the combs at a 45 angle to avoid trapping air underneath the comb teeth Air bubbles will inhibit polymerization and cause dents in the wells and a distorted pattern of protein bands 11 Remove the combs and rinse wells with 1x SDS electrophoresis buffer Remove the gels from the caster 12 Remove the gels from the caster and separate by carefully inserting a long razor blade or knife between the gel sandwiches A plastic wedge Hoefer s Wonder Wedge also works well 13 Clean the outside of each gel plate with running water to remove the residual poly merized and unpolymerized acrylamide 14 Overlay gels to be stored with 1x Tris Cl SDS pH 8 8 place in a resealable plastic bag and store at 4 C until needed up to 1 week SEPARATION OF PROTEINS ON GRADIENT GELS Gels that consist of a gradient of increasing polyacrylamide concentration resolve a much wider size range of proteins than standard single concentration gels see Critical Parameters and Troubleshooting The protein bands are also much sharper particularly in the low molecular weight range Unlike single concentration gels gradient gels separate proteins in a way that can be represented easily to give a linear plot from 10 to 200 kDa This facilitates molecular weight estimations The quantities given below provide separating
48. entation of filter and gel relative to the anode and cathode electrodes was used If the transfer efficiency using the tank sys tem appears to be low increase the transfer time or power Cooling using the unit s built in cooling cores is generally required for trans fers gt 1 hr At no time should the buffer tem perature go above 45 C Prolonged transfers gt 1 hr are not possible in semidry transfer units due to rapid buffer depletion Alternatively the transfer buffer can be modified to increase efficiency Adding SDS at a concentration of 0 1 to the transfer buffer improves the transfer of all proteins out of the gel particularly those above 60 to 90 kD in size Lowering the concentration of methanol will also improve the recovery of proteins from the gel These procedures are tradeoffs Methanol improves the binding of proteins to PVDF and nitrocellulose but at the same time hinders transfer With SDS present transfer efficiency is improved but the SDS can interfere with protein binding to the membrane Nylon and PVDF membranes are particularly sensitive to SDS interference If needed 0 01 to 0 02 SDS may be used in PVDF membrane transfer buffers Millipore 1990 SDS and methanol should not be used in the transfer buffer for nylon Peluso and Rosenberg 1987 Gel cross linking and thickness also have a profound effect on the transfer efficiency In general 0 5 to 0 75 mm thick gels will trans fer much more e
49. gel see Basic Protocol 1 steps 4 to 7 STAINING PROTEIN GELS WITH SILVER Silver staining is based on the selective reduction of silver ions at sites of the gel that contain proteins and other macromolecules The following protocol is adapted from that of Blum et al 1987 and involves pretreatment of a fixed polyacrylamide gel with thiosulfate step 6 followed by impregnation with silver nitrate step 9 and color development with formaldehyde at a high pH steps 12 and 13 Pretreatment with thiosulfate significantly increases the sensitivity and improves the contrast of protein staining In addition the presence of low concentrations of thiosulfate during color development reduces nonspecific staining of the gel surface The procedure is applicable for both nondenaturing UMIT 6 5 and SDS PAGE one or two dimensional unT 6 1 and UNIT 6 4 and yields dark brown to black protein bands on a clear background Materials Polyacrylamide gel containing protein s of interest see UNITS 6 1 6 4 amp 6 5 either unfixed or fixed and stained with Coomassie blue see Basic Protocol 1 and Alternate Protocol 1 Deionized water HPLC grade or Milli Q 50 v v ethanol in deionized water Fixative solution see recipe Thiosulfate solution see recipe Silver nitrate solution see recipe Developer solution see recipe 50 v v methanol 12 v v acetic acid in deionized water 50 v v methanol in water continued Current Prot
50. however the use of radioiodinated antibodies has been progressively replaced by nonradioactive detection with antibodies coupled to enzymes such as alkaline phosphatase or horseradish peroxidase see UNIT 16 5 The enzymes act on substrates which are converted to colored luminescent or fluorescent products Nonradioactive methods are just as sensitive as radioactive methods with the added advantage that they do not require the special precautions associated with the use of radioactivity Nonradioactive detection is nowadays the method of choice for visualizing immunoblotted proteins A disadvantage of nonradioactive methods is that they have a narrower linear range of detection which can be a problem in experiments that require accurate quantitation of protein levels Contributed by Juan S Bonifacino Current Protocols in Cell Biology 2002 6 0 1 6 0 3 Copyright 2002 by John Wiley amp Sons Inc Electrophoresis and Immunoblotting 6 0 1 Supplement 15 Introduction 6 0 2 Supplement 15 Radiolabeled proteins separated by electrophoresis or proteins detected by immunoblotting with radioiodinated antibodies or protein A can be visualized by autoradiography as described in UNIT 6 3 In this technique ionizing radiation emanating from the radionuclides impresses a photographic film The technique can be made more sensitive by the use of intensifying screens or scintillating compounds which emit light upon radiatio
51. hr 17 Disassemble the gel see Basic Protocol 1 steps 23 to 26 Proceed with detection of proteins PREPARING MULTIPLE GRADIENT MINIGELS Polyacrylamide gradients not only enhance the resolution of larger format gels but also greatly improve protein separation in the small format Casting gradient minigels one at a time is not generally feasible because of the small volumes used but multiple gel casters make it easy to cast several small gradient gels at one time The gels are cast from the bottom in multiple casters with the light acrylamide solution entering first This is the opposite of casting one gel at a time in which the heavy solution enters from the top of the gel sandwich and flows down to the bottom Current Protocols in Cell Biology Additional Materials also see Basic Protocol 2 Plug solution see recipe Additional reagents and equipment for preparing gradient gels see Alternate Protocol 5 Set up the system and prepare the gel solutions 1 Assemble minigel sandwiches in the multiple gel caster as described for single concentration minigels see Basic Protocol 2 steps 1 to 3 2 Set up the 30 ml gradient maker magnetic stirrer peristaltic pump optional and Tygon tubing as in Figure 6 1 3 Connect the outlet of the 30 ml gradient maker to the inlet at the base of the front faceplate of the caster The monomer solution will be introduced through the inlet at the bottom of the front faceplate of the ca
52. illumination or with a laser based gel scanner equipped with appropriate excitation source The enzymatic amplification step greatly enhances the signal allowing low nanogram detection of glycoproteins a sensi tivity on par with chemiluminescence detection methods Pro Q Glycoprotein Blot Stain kits with wheat germ agglutinin or with Griffonia simpliifolia lectin II GS II allow detection of N acetylglucosamine and sialic acid residues or terminal N acetylglu cosamine residues respectively The detection procedures for these lectins are quite similar to the concanavalin A method and the same fluorogenic substrate DDAO phos phate is used in the kits Materials Protein samples of interest PVDF membrane 50 methanol Wash solution II see recipe Blocking solution see recipe Pro Q Glycoprotein Blot Stain Kit with Concanavalin A Molecular Probes containing Concanavalin A alkaline phosphatase conjugate Con A AP stock solution see recipe DDAO phosphate stock solution see recipe Dimethylformamide DMF CandyCane glycoprotein molecular weight standards see recipe sufficient volume for 20 gel lanes Incubation buffer see recipe 10 mM Tris 1 mM MgCl pH 9 5 Polystyrene staining dishes e g weigh boat for minigel or larger container for larger gels Plastic wrap UV epi illumination and a digital or film camera or a laser equipped with a 633 nm helium neon laser or 635 nm diode laser source Additional reagents and equipm
53. in a change in A of the sample lanes when compared to the control The change in A can be calibrated to a known amount of protein loaded on the control lane This will be a relative value however since the transfer out of the gel and binding to the membrane is rarely 100 IMMUNOPROBING WITH DIRECTLY CONJUGATED SECONDARY ANTIBODY Immobilized proteins are probed with specific antibodies to identify and quantitate any antigens present The membrane is immersed in blocking buffer to fill all protein binding sites with a nonreactive protein or detergent Next it is placed in a solution containing the antibody directed against the antigen primary antibody The blot is washed and exposed to an enzyme antibody conjugate directed against the primary antibody secon dary antibody e g goat anti rabbit IgG Antigens are identified by chromogenic or luminescent visualization see Basic Protocol 3 and Alternate Protocol 4 of the anti gen primary antibody secondary antibody enzyme complex bound to the membrane Tween 20 is a common alternative to protein blocking agents when using nitrocellulose or PVDF filters Materials Membrane with transferred proteins see Basic Protocol 1 or Alternate Protocol 1 Blocking buffer appropriate for membrane and detection protocol see recipe Primary antibody specific for protein of interest TTBS nitrocellulose or PVDF or TBS nylon see APPENDIX 24 for recipes Secondary antibody conjugate horserad
54. increase the current for each additional gel that is connected to the power supply Two identical gels require double the current to achieve the same starting voltages and electrophoresis separation times power supply power supply power supply two gels connected two gels connected gels connected in series in parallel in parallel to one outlet to adjacent outlets to one outlet gel voltages are additive gel currents are additive gel currents are additive Figure 6 1 1 Series and parallel connections of gel tanks to power supply Current Protocols in Cell Biology Electrophoresis and Immunoblotting 6 1 3 Supplement 37 BASIC PROTOCOL 1 One Dimensional SDS PAGE 6 1 4 Supplement 37 Multiple gel apparatuses can also be connected to one pair of outlets on a power supply This is useful with older power supplies that have a limited number of outlets When connecting several gel units to one outlet make certain the connections between the units are shielded and protected from moisture The gels can be connected in parallel or in series Fig 6 1 1 When gels are connected through the same outlet in parallel the principle of additive currents is the same as for gels connected through different outlets in parallel In the case of two or more gels running off the same outlet in series the current is the same for every gel If 10 mA is displayed by the power supply meter for example each gel in s
55. is separated in the same gel as the protein sample containing the unknown The standards are used to create a standard curve of relative mobility versus size or molecular mass Although digital image analysis has greatly simplified calculating the mass of an unknown protein separated by electrophoresis manual assessments of molecular mass are useful and use the same basic calculations 1 Calculate the relative mobility Rf using following formula Rr distance migrated by protein distance migrated by marker Placing a molecular mass R mobility acetate overlay calculator Fig 6 1 5 on the gel is a quick way to determine Ry Simply align the top and bottom of the overlay with the top of the gel and the dye front respectively to get a read out of Ry Figure 6 1 5 Example of an R calculator This sheet is copied to transparency film using a paper copier and used as an overlay on the gel When the transparency is placed on top of the gel so that the top of the gel aligns with the top of the calculator and the dye front aligns with the bottom of the calculator the R can be read directly off the overlay Note that the calculator accommodates a range of gel lengths The overlay should be copied at a 1 1 ratio so that the centimeter scale remains accurate However as long as the overlay can fit the top and bottom of the gel the R numbers will be accurate 6 1 28 Supplement 37 Current Protocols in Cell Biology 2 Plot
56. lines J Biol Chem 54 7961 7977 Garrels J I 1989 The QUEST system for quantita tive analysis of two dimensional gels J Biol Chem 264 5269 5282 Garrels J I and Franza Jr B R 1989 The REF52 protein database Methods of database construc tion and analysis using the QUEST system and characterizations of protein patterns from prolif erating and quiescent REF52 cells J Biol Chem 264 5283 5298 Goverman J and Lewis K 1991 Separation of disulfide bonded polypeptides using two di mensional diagonal gel electrophoresis Meth ods 3 125 127 Hochstrasser D F Harrington M C Hochstrasser A C Miller M J and Merril C R 1988 Meth ods for increasing the resolution of two dimen sional protein electrophoresis Anal Biochem 173 424 435 O Farrell P H 1975 High resolution two dimen sional electrophoresis of proteins J Biol Chem 250 4007 4021 Strahler J R and Hanash S M 1991 Immobilized pH gradients Analytical and preparative use Methods 3 109 114 Young D A Voris B P Maytin E V and Colbert R A 1983 Very high resolution two dimen sional electrophoretic separation of proteins on giant gels Methods Enzymol 91 190 214 Key Reference Hochstrasser et al 1988 See above Discusses methods for improving and troubleshoot ing two dimensional separation Contributed by Sandra Harper Jacek Mozdzanowski and David Speicher The Wistar Institute Philadelphia Pen
57. lt 1 5 mm in diameter can be prepared by the same method but extrusion of the thinner agarose gel without breaking is more difficult The protocol supplies molecular weight markers containing 2 5 ug of each standard suitable for Coomassie blue staining or 0 25 ug of each standard for silver staining Materials Molecular weight standards Table 6 1 2 1x SDS sample buffer UNIT 6 1 2 w v agarose see recipe Boiling water bath Glass tubes 3 mm inner diameter Plastic or metal tray 1 Prepare 3 ml molecular weight standards in 1x SDS sample buffer using 250 ug of each standard The stated amount is appropriate for Coomassie blue staining of gels If silver staining is planned use 25 ug of each standard 2 Mix the standards with 2 ml of 2 w v agarose melted in a boiling water bath Oo Prepare clean glass tubes by wrapping one end with Parafilm Pour the hot mixture into the tubes and let the agarose solidify Carefully extrude the agarose from the tubes Cut agarose rods into 5 mm pieces using a razor blade Freeze all pieces separately on a plastic or metal tray using dry ice NHN NW A Collect frozen pieces in a plastic bottle and store at 80 C The standards may be stored 21 year Current Protocols in Cell Biology DIAGONAL GEL ELECTROPHORESIS NONREDUCING REDUCING GELS Protein subunit compositions and cross linked protein complexes can be analyzed by two dimensional gel electrophoresis using sep
58. marking orientation as in step 12 of Basic Protocol 1 Proceed with staining and immunoprobing see Basic Protocol 1 steps 13 and 14 BLOTTING OF STAINED GELS Gels stained with Coomassie blue R250 can be effectively immunoblotted by the following procedure based on Perides et al 1986 and Dionisi et al 1995 Briefly the stained gel is soaked in a series of solutions designed to increase the solubility of the proteins after staining and fixation After transfer the membranes are treated with 45 or 100 methanol to decrease the Coomassie blue bound to the membrane prior to processing for chromogenic development For chemiluminescent development removal of the Coomassie blue is generally not needed Materials Destained gel containing proteins of interest 25 mM Tris base 192 mM glycine 1 SDS 25 mM Tris base 192 mM glycine 0 1 SDS Current Protocols in Cell Biology 1 Soak destained gel containing proteins of interest in distilled water for 15 min 2 Equilibrate gel with 25 mM Tris base 192 mM glycine 1 SDS for 1 hr with gentle agitation 3 Transfer gel to 25 mM Tris base 192 mM glycine 0 1 SDS and equilibrate 30 min with gentle agitation To increase transfer efficiency of larger proteins the gel should be transferred to the above solution with 6 M urea for an additional 30 min 4 Proceed with transfer see Basic Protocol 1 steps 2 to 12 For the most efficient transfer and binding to the membrane the transfer bu
59. maximum current of 0 8 mA cm of gel area For a typical minigel 8 x 10 cm and standard sized gel 14 x 14 cm this means 60 and 200 mA respectively 2 Prepare transfer membrane see Basic Protocol 1 step 6 3 Disassemble gel sandwich Remove and discard stacking gel Equilibration of the separating gel with transfer buffer is not normally required for semidry blotting but it may improve transfer in some cases 4 Place three sheets of filter paper saturated with transfer buffer on the anode Fig 6 2 2 Most transfer units are designed so that negatively charged proteins move downward toward either a platinum or graphite positive electrode anode CAPS transfer buffer pH 10 5 see recipe for transfer buffer can be used in place of the Tris glycine methanol transfer buffer of Basic Protocol 1 CAPS buffer should be used if the protein is to be sequenced right on the membrane Moos 1992 as glycine will interfere with this procedure The filter paper should be cut to the exact size of the gel This forces the current to flow only through the gel and not through overlapping filter paper Some manufacturers e g Amersham Pharmacia Biotech recommend placing a Mylar mask on the lower platinum anode With an opening that is slightly less than the size of the gel the mask forces the current to flow through the gel and not the surrounding electrode area during transfer 5 Place equilibrated transfer membrane on top of fil
60. membrane 6 Transfer membrane to fresh plastic bag containing secondary antibody solution Incubate 30 min at room temperature with slow rocking then wash as in step 4 When using plastic incubation trays see step 3 annotations for proper antibody solution volumes 7 While membrane is being incubated with secondary antibody prepare avidin biotin HRPO or AP complex Mix two drops Vectastain reagent A and two drops reagent B into 10 ml TTBS nitrocellulose or PVDF or TBS nylon Incubate 30 min at room temperature then further dilute to 50 ml with TTBS or TBS Diluting the A and B reagents to 50 ml expands the amount of membrane that can be probed without greatly affecting sensitivity Sodium azide is a peroxidase inhibitor and should not be used as a preservative Casein nonfat dry milk serum and some grades of BSA may interfere with the formation of the avidin biotin complex and should not be used in the presence of avidin or biotin reagents Gillespie and Hudspeth 1991 Vector Labs 8 Transfer membrane to avidin biotin enzyme solution Incubate 30 min at room temperature with slow rocking then wash over a 30 min span as in step 4 9 Develop membrane according to the appropriate visualization protocol see Basic Protocol 3 or Alternate Protocol 4 VISUALIZATION WITH CHROMOGENIC SUBSTRATES Bound antigens are typically visualized with chromogenic substrates The substrates 4CN DAB NiCl and TMB are commonly used with hor
61. of a first dimension BN PAGE by two dimensional BN SDS PAGE in order to prove that the de tected protein indeed is the protein of interest and not a cross reactivity of the antibody Performing a successful NAMOS assay critically depends on the quality of the anti bodies used to shift the protein complex of interest Swamy et al 2007 Several prob lems could arise First protein complexes clus tered by antibodies can be very large thus use Alternate Protocol 1 to disassemble these aggregates to efficiently transfer them to the blotting membrane Second some antibodies aggregate with themselves thus producing a ladder like shift pattern Swamy et al 2007 With these antibodies a continuous increase in antibody concentration leads to a constant gen eration of new larger bands while smaller ones disappear Thus the stoichiometry of a mul tiprotein complex cannot be determined with this type of antibody however it is possible to determine whether all complexes contain the subunit in question Use a fresh prepara tion of antibody prepare Fab fragments or re move antibody aggregates by ultracentrifuga tion Third it is possible that a given antibody cannot bind to all identical subunits of a mul tiprotein complex probably because the com plex is not symmetric rendering the epitope accessible in one copy of the subunit but not in its neighboring copy Likewise if the two epi topes are very close within the complex
62. of the proteins being separated Under nondenaturing conditions the biological activity of a protein will be maintained Comparison of reducing and nonreducing denaturing gels can also provide valuable in formation about the number of disulfide cross linked subunits in a protein complex If the subunits are held together by disulfide link ages the protein will separate in denaturing gels as a complex or as smaller sized sub units under nonreducing or reducing condi tions respectively However proteins sepa rated on nonreducing denaturing gels appear more diffuse and exhibit less overall resolution than those separated on reducing gels Gradient gels provide superior protein band sharpness and resolve a larger size range of proteins making them ideal for most types of experiments in spite of being more dif ficult to prepare Molecular weight calcula tions are simplified because of the extended linear relationship between size and protein Current Protocols in Cell Biology position in the gel Increased band sharpness of both high and low molecular weight pro teins on the same gel greatly simplifies survey experiments such as gene expression stud ies where the characteristics of the responsive protein are not known Furthermore the in creased resolution dramatically improves au toradiographic analysis Preparation of gradi ent gels is straightforward although practice with gradient solutions containing dye is rec o
63. on Electroblots 6 8 14 Supplement 16 Anticipated Results The performance characteristics of the Pro Q Emerald 300 Glycoprotein Gel Stain Kit Pro Q Emerald 300 Glycoprotein Blot Stain Kit and Pro Q Glycoprotein Detection Kit with Concanavalin A are summarized in Table 6 8 1 and contrasted with alternative glycoprotein detection technologies The fluorescence based methods permit detection of low nanogram amounts of glycoprotein with a dy namic range of quantitation that encompasses three orders of magnitude of glycoprotein abundance Pro Q Emerald 300 dye may be used to detect a variety of glycoconjugates in addition to glycoproteins such as bacterial lipopolysaccharides LPS and glycogen De tection sensitivity for chondroitin 4 sulfate however is 3000 fold poorer than glycogen or LPS with limits of detection in the vicinity of 16 ug of applied material This is not unex pected as glycosaminoglycans such as chon droitin sulfate hyaluronic acid and keratan sulfate are known to stain poorly by conven tional PAS procedures Concanavalin A specifi cally binds to nonsubstituted and 2 O substi tuted o mannosyl residues and thus detects fewer glycoproteins than the Pro Q Emerald 300 dye For example 1 acid glycoprotein is not detected by concanavalin A Similarly gly coproteins such as ovomucoid 28 kDa and ovotransferrin 76 kDa are not effectively de tected by concanavalin A The differences in staining
64. optimal pH samples may precipitate in the gel at the loading position Current Protocols in Cell Biology 14 Position one sample cup above each gel strip and push down to ensure good contact between the bottom of the sample cup and the gel strip Make sure the gel strips have not shifted position 15 Pour 70 to 80 ml of DryStrip cover fluid into the tray it will cover the gels If oil leaks into the sample cups adjust the cups to stop leakage When there is no leakage into the sample cups add enough cover fluid to the tray to completely cover the sample cups 150 ml 16 Pipet protein samples in lysis buffer into the sample cups by underlaying The sample should sink to the bottom of the cup Check for leakage of the sample out of the sample cup Samples should either be lyophilized and then solubilized in lysis buffer or diluted 9 parts lysis buffer to 1 part sample The maximum volume each sample cup holds is 100 ul The complexity of the sample the sample solubility at the loading concentration and pH used the thickness of the second dimension gel and the detection method to be employed should be considered when deciding how much protein to load As a starting reference typical loading ranges for 1 0 to 1 5 mm thick 18 cm x 18 cm gels would be 5 to 20 ng per major spot for silver staining and 1 to 5 ug per major spot for Coomassie blue staining When very complex samples are used such as whole cell extracts total protein loa
65. or other information that was entered at the time of image capture More powerful database products are also available that can perform complex searches on data generated during the analysis For an example a search on a two di mensional database might include finding pro teins exhibiting a specific expression profile and having a molecular weight gt 20 kDa with a pI between 3 and 5 or 8 and 10 with an amount lt 50 ng in a series of experiments conducted lt 1 year ago Such searches can quickly target potentially interesting molecules for further analysis With the increasing ease of transferring data through the Internet as well as local and wide area networks it has become practical to quickly find and examine data from distant locations Of course great care must be taken to ensure that similar experimental conditions are employed as otherwise the results will be difficult to compare In this manner it is some times possible to dramatically increase the sam ple size and statistical accuracy as well as the probability of detecting rare events In addition if one data set is more completely charac terized this extra information can be extracted and applied to the other data set For example if there is a band in common in two databases and there is sequence information for it in one database that sequence information can be added to the other database Currently most public electrophoresis database sites are two dimensional
66. protein assay kit Pierce DNase and RNase solution see recipe 20 w v SDS 4PPENDIX 2E 2 Mercaptoethanol Urea ultrapure Lysis buffer see recipe 50 ml centrifuge tube Centrifuge with rotor e g Beckman JS 4 2 4 C 1 to 2 ml cryovials Microcentrifuge 4 C Sonicator with microtip 0 2 um microcentrifuge filter units e g Millipore Ultrafree MC filter units Harvest and wash the cells 1 Place cell culture flasks containing cells of interest on ice 2 Rapidly wash cells three times with 2 to 6 ml PBS I buffer Keep the flasks on ice The required volume of PBS I buffer depends on the flask size For example use 2 ml for a 25 cm tissue culture flask and 6 ml for a 75 cm tissue culture flask 3 Add 2 to 6 ml PBS I buffer to the flask scrape the cells using a cell scraper and transfer the suspension to a 50 ml centrifuge tube Repeat this step with another 2 to 6 ml buffer to ensure complete transfer of the cells Cells grown in suspension are washed in an analogous manner using repetitive centrifu gation 4 Collect the cells by centrifuging 15 min at 2600 x g 3000 rpm in Beckman JS 4 2 rotor 4 C 5 Discard the supernatant and resuspend the cells in a small volume of PBS I buffer 6 Transfer the cell suspension to a labeled cryovial The weight of the empty cryovial can be determined prior to use if the wet weight of the cell pellet is desired as a reference value rather than cell number radio
67. protein databases A list with links Current Protocols in Cell Biology to many of these Internet database sites can be found at http www lmmb ncifcrf gov EP ta ble2Ddatabases html With biological questions becoming more complicated and the answers to the questions often requiring information from a variety of sources it is becoming increasingly important to be able to move easily between information sources A relational type of database can help achieve this Unlike a conventional database with a fixed arrangement of data relational databases have links between related files that allow for easy movement from one file to an other Another approach to interconnecting electrophoresis data with data from other sources is to generate a series of hypertext links between data sets similar to what occurs on the Internet Selecting a specific link moves the search to the related network site and the related information Regardless of the method the end goal is similar An example of what is possible a researcher selects a protein spot on a two di mensional gel image which triggers accessing of related information on this protein The pro tein sequence is accessed from mass spectros copy analysis of the spot on a separate gel The sequence of the gene and the cDNA that gen erated the protein is retrieved The expression pattern of the gene in various tissues and con ditions as well as information on similar genes in other organisms
68. range Basic Protocol 1 is based on use of 3 mm first dimension isoelectric focusing gels and 1 5 mm second dimension gels using the Bio Rad two dimensional gel apparatus Protean II xi2D The method can be easily adapted to equipment from other suppliers or to different sized gels by adjusting the quantities of reagents used The protocol uses 8 M urea and Triton X 100 as solubilizing agents Solubilization of the protein sample applied to the gel as well as maintenance of solubility during electric focusing are the most Current Protocols in Cell Biology critical factors influencing the quality of sepa ration in the first dimension The most common modification to Triton X 100 based proce dures is addition of 3 3 cholamidopropyl di methylammonio 1 propanesulfonate CHAPS to the gel and solubilizing buffer mixtures Addition of SDS to complex samples such as tissue or cell extracts can also enhance reproducible solubilization of the largest pos sible subset of proteins Although SDS is charged in the presence of Triton X 100 it is separated from proteins during focusing and migrates to the acidic end of the gel Regardless of the method used to maintain solubility some proteins especially those gt 100 kDa tend to precipitate at pH values approaching their isoelec tric point and thus produce horizontal smears on the final second dimension gel Basic Protocol requires several modifica tions for successful separation o
69. room temperature Perform two final washes in wash solution II for 5 min each at room temperature 10 Dilute the DDAO phosphate stock solution 1000 fold into 10 mM Tris 1 mM MgCl pH 9 5 for a final concentration of 1 25 uM Approximately I ml of the DDAO phosphate staining solution will be needed for an 8x 10 cm blot Note that DDAO phosphate is unstable when stored at room temperature as an aqueous solution Always make up the DDAO phosphate staining solution just prior to use Incubate the blot in freshly prepared DDAO phosphate staining solution The staining step may be performed either face up or face down depending on the configuration of the imaging instrumentation being used If using UV epi illumination or a laser scanner with a light source that illuminates from above the imaging bed stain the blot face up For laser scanners with light sources that illuminate the blot from below the imaging bed stain the blot face down Using powder free gloves cut a piece of plastic wrap to the size of the blot For face up staining place the blot on the plastic wrap and pipet 1 ml of DDAO phosphate staining solution onto the blot For face down staining pipet 1 ml of the DDAO Current Protocols in Cell Biology Electrophoresis and Immunoblotting 6 8 9 Supplement 16 Fluorescence Detection of Glycoproteins in Gels and on Electroblots 6 8 10 Supplement 16 13 14 phosphate staining solution onto
70. room temperature REAGENTS AND SOLUTIONS Use deionized or distilled water in all recipes and protocol steps For common stock solutions see APPENDIX 2A for suppliers see SUPPLIERS APPENDIX Blocking solution 50 mM Tris Cl pH 7 5 APPENDIX 2A 150 mM NaCl 0 2 v v Tween 20 0 25 w v Mowiol 4 88 Calbiochem or VWR Store up to 6 months at room temperature The use of Mowiol 4 88 in the Blocking buffer is not essential but does decrease background staining and improves detection sensitivity As an alternative to Mowiol 4 88 0 5 w v bovine serum albumin or 4 w v gelatin high purity e g TopBlock from Juro Supply may be used CandyCane molecular weight standards For a standard lane on an 8 x 10 cm gel dilute 0 5 ul of the standards with 7 5 ul of 2x sample buffer see recipe and vortex This will result in 250 ng of each protein per lane a sufficient amount for detection of the glycoproteins by the Pro Q Emerald 300 stain For large 16 x 18 cm gels double the amount of standard and buffer used Store up to 6 months at room temperature Con A AP stock solution Prepare a 2 mg ml stock solution of Con A AP by dissolving the vial contents in 250 ul deionized water and add 2 mM sodium azide The stock solution is stable for at least 6 months when stored undiluted at 4 C Do not freeze Current Protocols in Cell Biology DDAO phosphate stock solution Add 200 ul DMF to the vial containing 250 ug DDAO phosphate
71. standard electroblotting procedures UNIT 6 2 The use of nitrocellulose membranes is not recommended Optional Stain blots with SYPRO Ruby protein blot stain at this point to visualize the total protein pattern and to verify that the blotting procedure was successful Follow the staining procedure described in Alternate Protocol steps 14 to 17 Total protein staining must be performed prior to lectin blotting as the blocking mixture will produce very high background on the blot Since SYPRO Ruby protein blot stain is washed off during the subsequent lectin blotting process it is important to document staining results before continuing with the procedure If the PVDF blot is dry briefly hydrate in 50 methanol and incubate in wash solution II for 10 min at room temperature Repeat the wash step for a total of three washes Visualize glycoproteins 6 7 11 12 Incubate the blot in blocking solution for 1 to 2 hr at room temperature Briefly pellet any potential protein aggregates in the Con A AP stock solution by microcentrifugation Using the supernatant only dilute the Con A AP stock solution 2000 fold by adding 5 ul to 10 ml of incubation buffer for a final concentration of 1 ug ml Remove the blocking solution that the blot is immersed in and incubate the blot with Con A AP solution for 1 hr at room temperature Remove the diluted Con A AP solution and wash the blot in blocking solution four times for 10 min each at
72. strips Separate antigens on a preparative gel i e a single large sample well and immunoblot the entire gel Cut 2 to 4 mm strips by hand or with a membrane cutter Schleicher and Schuell Inotech and incubate individual strips in a set of serial dilutions of primary antibody The correct dilution should give low background and high specificity Fig 6 2 3 3 Open bag remove blocking buffer and add enough primary antibody solution to cover membrane Incubate 30 min at room temperature with gentle rocking Usually 5 ml diluted primary antibody solution is sufficient for two to three membranes 14 x 14 cm size Incubation time may vary depending on conjugate used When using plastic trays the primary and secondary antibody solution volume should be increased to 25 to 50 ml For membrane strips incubation trays with individual slots are recommended Typically 0 5 to 1 ml solution slot is needed 4 Remove membrane from bag and place in plastic box Wash membrane three times over a 15 min span in TTBS nitrocellulose or PVDF or TBS nylon Add enough TTBS or TBS to fully cover the membrane e g 5 to 10 ml strip or 25 to 50 ml whole membrane Current Protocols in Cell Biology 5 Prepare biotinylated secondary antibody solution by diluting two drops biotinylated antibody with 50 to 100 ml TTBS nitrocellulose or PVDF or TBS nylon This dilution gives both high sensitivity and enough volume to easily cover a large 14 x 14 cm
73. syringe 28 Remove one gel tube at a time from the chamber 29 Using a 10 ml syringe equipped with a blunt needle slowly and carefully inject water between the gel and glass tube Start from the bottom of the tube then repeat the procedure from the top The gel should slide out of the glass tube Current Protocols in Cell Biology It is convenient to let the gel slide from the glass tube onto a metal or plastic scoop which facilitates transfer of the gel into a storage vial It is relatively easy to break the gel during extrusion and practicing on several unused gels is recommended To extrude smaller di ameter gels use water pressure generated by a syringe connected to the gel tube with Tygon tubing If clean unscratched glass tubes are used extrusion should be easy 30 Using the scoop slide the gel into a 4 5 ml cryovial containing 3 ml equilibration buffer and 50 ul 2 mercaptoethanol Close the vial incubate exactly 5 min at room temperature then freeze by placing the tube horizontally on top of dry ice pellets Do not move or agitate the tube while the sample is freezing The IEF gels may be run on a second dimension gel immediately see Basic Protocol 3 or can be stored at 80 C for many weeks Even when the second dimension is to be run immediately extruded gels should be frozen after a carefully controlled incubation time at room temperature such as the 5 min cited above for 3 mm i d gels to minimize diffusion of
74. that of generating a pseudoimage of electrophoresis results through the use of a finish line type of detection system This is comprised of a light source positioned at the bottom of the gel i e the end opposite of the site of sample loading and light detectors positioned next to each lane to detect the trans mitted light or emitted fluorescence A lane trace is generated using time on the x axis and light intensity on the y axis The pseudoimage is then generated from this data Sutherland et al 1987 Capture Process Prior to image capture electrophoretic sepa ration and any visualization steps are per formed To calibrate the separation process standards are usually run at the edges of the gel and often at internal positions If quantitation of specific proteins or nucleic acids is to be performed a dilution series of standards with similar properties to the experimental samples should also be included After separation the protein or nucleic acid is visualized if neces sary Visualization can include binding of a fluorochrome or chromophore such as Coomassie blue precipitation of metal ions such as copper silver or gold enzymatic reac tions and exposure of film or phosphor screens to radiant sources These methods can be grouped based on the type of detection into optical density fluorescence chemilumines cence and radioactivity The suitability of popular detection devices with these methods is described in Tab
75. the 50x Pro Q Emerald 300 concentrate reagent 50 fold into Pro Q Emerald 300 dilution buffer For example dilute 500 ul of 50x Pro Q Emerald 300 concentrate reagent into 25 ml of dilution buffer to make enough staining solution for one 8 x 10 cm gel Current Protocols in Cell Biology 9 Incubate the blot in the dark in 25 ml Pro Q Emerald 300 staining solution step 8 while gently agitating for 90 to 120 min room temperature The signal can be seen after 20 min and maximum sensitivity is reached at 120 min Staining overnight is not recommended 10 Wash the blot with 25 ml wash solution for 15 min at room temperature Repeat this wash one additional time Do not leave the blot in wash solution for gt 2 hr as the staining signal will start to decrease 11 Allow the membrane to air dry 12 Visualize the stain using a standard 300 nm UV epi illuminator The Pro Q Emerald 300 stain has an excitation maximum at 280 nm and an emission maximum near 530 nm A UV transilluminator may also be used to visualize the glycoproteins but this results in poorer detection sensitivity 13 Document results before proceeding to the next step using a photographic camera or CCD camera with the appropriate filters to obtain the greatest sensitivity Pro Q Emerald dye signal will fade after SYPRO Ruby dye staining Visualize total protein 14 In order to counter stain non glycosylated proteins in the sample pour the SYPRO Ruby protein blot s
76. the GelBond and the opposing glass surface to remove any particulate material e g dust Sequencing gel spacers can be easily adapted First cut the spacers slightly longer than the length of the gel plate Position a spacer along each edge of the glass plate and assemble the gel sandwich clamping in place With a razor blade trim the excess spacer at top and bottom to get a reusable spacer exactly the size of the plate Prepare and pour the separating and stacking gels see Basic Protocol 1 steps 3 to 9 In place of the Teflon comb insert a square well sequencing comb cut to fit within the gel sandwich Allow the stacking gel to polymerize 30 to 45 min at room temperature Less solution is needed for ultrathin gels For example a 0 5 mm thick gel requires 33 less gel solution than a 0 75 mm gel Prepare the sample and load the gel see Basic Protocol 1 steps 12 to 20 When preparing protein samples for ultrathin gels 3 to 4 ulat 5 ug protein ul is required for Coomassie blue R 250 staining whereas 10 fold less is needed for silver staining Run the gel see Basic Protocol 1 steps 21 and 22 except conduct the electrophoresis at 7 mA gel 0 25 mm thick gels or 14 mA gel 0 5 mm thick gels for 4 to 5 hr When the separation is complete disassemble the unit and remove the gel see Basic Protocol 1 steps 23 to 26 with the GelBond still attached With a gloved hand wash away the adhesive material from the bac
77. the gel are potentially radioactive Carefully pour off the water and position the gel in the center of the glass dish Be sure that any excess water is drained Place a sheet of Whatman 3MM filter paper at least 1 to 2 cm larger than the gel over the gel The gel will stick to the filter paper which will allow it to be lifted and turned over with the gel side facing up Cover gel with plastic wrap Smooth the wrap with a piece of tissue paper to remove any air bubbles or wrinkles Place a piece of filter paper on the gel support of the gel dryer to prevent contamination of the dryer by radioactivity Place the filter paper gel plastic wrap sandwich on the filter paper in the gel dryer with the plastic sheet facing up Position the rubber sealing gasket of the gel dryer over the gel Set the appropriate heat setting on the gel dryer normally 80 C 60 C if the gel contains a fluor Apply the vacuum and allow the gel to dry typically 2 hr for a gel of 1 mm thickness Current Protocols in Cell Biology Removing the gel before it is completely dry can lead to cracking it is therefore not a good idea to rush the drying process A rough indication of whether the gel is dry can be obtained by feeling the gel under the sealing gasket If the gel is dry it should be warm over the entire surface 9 Remove gel from dryer and proceed with autoradiography see Basic Protocol USE OF INTENSIFYING SCREENS Intensif
78. the nominal resolution due to the need for two detectors for every resolvable object one for the object and one for the separation space and the effects of optical resolution Figure 6 9 2 demonstrates how spatial resolution can affect detection of objects The 42 um resolution image allows detection of closely spaced bands the 168 um resolution image detects fewer bands and the 840 um resolution image detects only major bands For instruments with on line detection systems a pseudo spatial resolution is often reported in units of time from the start of the separation or the time interval between two objects crossing the detection path Intensity resolution is the ability to identify small changes in intensity It is a function of both the dynamic range of a detector and the number of potential values that detector can report Greater dynamic range decreases the intensity resolution of a given detector The number of potential values a detector reports is Current Protocols in Cell Biology described by its bit depth An 8 bit detector can report 256 28 different possible values while a 12 bit detector can report 4 096 2 values and a 16 bit detector can report 65 536 2 values The higher the bit depth the greater the intensity resolution Technique dependent resolution directly af fects the spatial and intensity resolution Elec trophoretic separation techniques that generate overlapping objects or that have object sepa
79. they will be difficult to remove from the caster The gels can be stored tightly wrapped in plastic wrap with the combs left in place inside a sealable bag to prevent drying for 1 week Without the stacking gel the separating gel can be stored for 2 to 3 weeks Keep gels moist with 1 x Tris Cl SDS pH 8 8 at 4 C Do not store gels in the multiple caster Prepare the sample load the gel and conduct electrophoresis 12 Remove the combs and rinse the sample wells with 1x SDS electrophoresis buffer Place a line indicating the bottom of each well on the front glass plate with a marker 13 Fill the upper and lower buffer chambers with 1x SDS electrophoresis buffer The upper chamber should be filled to 1 to 2 cm over the notched plate 14 Prepare the protein sample and protein standards mixture see Basic Protocol 1 step 12 15 Load the sample using a micropipet Insert the pipet tip through the upper buffer and into the well The mark on the glass plate will act as a guide Dispense the sample into the well For a complex mixture 20 to 25 ug protein in 10 ul SDS sample buffer will give a strongly stained Coomassie blue pattern Much smaller amounts 1 to 5 ug are required for highly purified proteins and a 10 to 100 fold smaller amount of protein in the same volume e g 10 ul is required for silver staining 16 Electrophorese samples at 10 to 15 mA per 0 75 mm gel until the dye front reaches the bottom of the gel 1 to 1 5
80. this purpose Standard laboratory light boxes may also be used 2 Cover gel or filter with plastic wrap to protect the exposure cassette Place wrapped gel or filter in the PhosphorImager cassette and close to begin exposure The gel does not have to be dried for this procedure The phosphor screen is affixed to the lid of the cassette Exposure times are typically one tenth of the time required for film exposure 3 After exposure slide the screen face down into the PhosphorImager system 4 Select the scanning area using the software supplied with the PhosphorImager and start scanning The blue light emitted during scanning is collected to produce the latent image 5 Analyze and quantitate the image using the software provided 6 Erase the phosphor screen by exposing it to visible light as in step 1 COMMENTARY Background Information The ability to detect radiolabeled proteins is critical to many studies in cell biology A vari ety of labeling methods are described through out this manual many of which are used to follow protein purification protein processing or the movement of proteins within the cell More often than not detection of radiolabeled proteins is coupled with the resolving power of SDS polyacrylamide gel electrophoresis SDS PAGE unit 6 1 Radiolabeled proteins sepa rated on gels can be used directly to obtain an autoradiographic image Alternatively pro teins separated by SDS PAGE are frequen
81. to get acceptable performance One example of a secondary process is to discard spots with sizes below a set minimum or above a set maximum Another is to analyze spots that are oval for possible splitting into two spots Even after secondary processing it is likely that a small amount of manual editing will be necessary When manually editing an image care must be taken to use as objective criteria as possible especially if two or more images are to be matched and spot volumes compared Characterization In atwo dimensional system determination of protein or nucleic acid mobility is compli cated by the fact that there are two mobilities to account for and that the second dimension separation tends to make estimation of the sepa ration which occurred in the first dimension more difficult One method for dealing with this is to have a series of markers in the sample that after both separations are completed are evenly distributed within the gel and image It is also possible to estimate separation characteristics from calibration points located at the periphery of the gel For example distance measurements can be used to pass calibration data from the first dimension separation and standards can be separated at the ends of the gel to calibrate for mobility in the second dimension Regard less of the method used in many instances a series of related images will be examined and similar spots in each image will be matched When thi
82. to the recipes in Table 6 5 1 Table 6 5 2 Table 6 5 3 or Table 6 5 4 adding the ammonium persulfate and TEMED just before use Deaeration of the solution before the polymerization catalysts are added will speed polymerization by removing inhibitory oxygen but this is not generally required The pH used depends on many factors The most important are the pI values of both the protein of interest and any contaminants as well as protein mobility and protein solubility Deter mining which pH and thus which buffer system to use is largely empirical However extremes of pH lt 4 0 and gt 9 0 can lead to denaturation and should be avoided Prior knowledge of the pI of a protein UNITS 6 4 allows determination of the net charge under Table 6 5 1 Recipes for Acetic Acid Nondenaturing Polyacrylamide Gels pH Range 3 7 to 5 6 Final acrylamide concentration in gel 5 7 5 10 12 5 15 17 5 20 Stock solution 30 acrylamide 0 8 6 7 10 13 3 16 8 20 23 32 26 6 bisacrylamide 300 mM sodium sulfite 0 4 0 4 0 4 0 4 0 4 0 4 0 4 4x acetic acid gel buffer 10 10 10 10 10 10 10 H O 22 58 19 28 15 98 1248 928 5 96 2 68 10 w v ammonium 0 3 0 3 0 3 0 3 0 3 0 3 0 3 persulfate TEMED 0 02 0 02 0 002 0 02 0 02 0 02 0 02 Preparation of gel In a 75 ml side arm flask mix 30 acrylamide 0 8 bisacrylamide solution see Table 6 1 1 300 mM sodium sulfite 4x acetic acid gel buffer see Reagents and Solutions and H O If d
83. two antibody molecules might not be able to bind simultaneously due to steric hindrance This type of antibody gives an underestimate of the actual stoichiometry Swamy et al 2007 In conclusion not all antibodies result in the number of shifts that correspond to the num ber of subunit copies present in the protein complex Thus it is recommended to use sev eral independent antibodies per subunit and the Fab fragments of those antibodies Anticipated Results In BN PAGE native protein and multipro tein complexes are separated according to their size Thus using two dimensional BN SDS PAGE protein complexes can be identified and characterized in terms of their relative abun dance subunit composition and size Since BN PAGE is only the separation technique the sensitivity of detection depends on the detec tion method used Furthermore the NAMOS assay allows the determination of the stoi chiometries even from nonpurified complexes Typical results are illustrated in Figures 6 10 1 and 6 10 2 Comparison of two or more sam ples is possible if the pouring and running of the gels was done in similar way In combi nation with modern mass spectroscopy BN PAGE with its high resolution properties is an excellent choice to identify novel protein complexes from any biological source Time Considerations Preparation of the sample as described in the Support Protocol takes 6 hr or overnight If purified proteins or organel
84. vol of 50 ethanol in deionized water Incubate 20 min with gentle agitation 4 Replace the solution with another 10 gel vol of 50 v v ethanol in deionized water Incubate 20 min with gentle agitation Repeat this step once total of three pretreat ment steps The plastic container used in steps 3 and 4 can be reused in step 16 Discard the ethanol solution and rinse the container briefly with deionized water 5 Transfer the gel to a clean plastic container having 10 gel vol deionized water and incubate 5 to 10 min with gentle agitation 6 Discard the water and add 10 gel vol thiosulfate solution Incubate for exactly 1 min with gentle agitation Incubations longer than 1 min will result in increased background staining 7 Tranfer the gel to a clean plastic container having 10 gel vol deionized water and incubate exactly 20 sec 8 Change the water in the container and incubate exactly 20 sec Perform this step twice for a total of three The plastic container used in steps 7 and 8 can be reused in steps 10 and 11 but should first be rinsed thoroughly with deionized water Stain the gel 9 Transfer the gel to a clean plastic container containing 5 to 10 gel vol silver nitrate solution Cover the container with aluminum foil and incubate 20 min with gentle agitation Light exposure causes the reduction of silver ions and may therefore result in increased background staining Current Protocols in Cell Biology 10 Tran
85. wells because the glycerol added to the sample buffer gives the solution a greater density than the electrophoresis buffer To keep bands tight hold the tip of the needle near the bottom of the well and load the samples slowly The bromphenol blue in the sample buffer makes sample application easy to follow visually Fill the remainder of the upper buffer chamber with additional 1 x SDS electrophore sis buffer so that the upper platinum electrode is completely covered Do this slowly so that samples are not swept into adjacent wells Current Protocols in Cell Biology Electrophoresis and Immunoblotting re 6 1 9 Supplement 37 ALTERNATE PROTOCOL 1 One Dimensional SDS PAGE 6 1 10 Supplement 37 Run the gel 21 Connect the power supply to the cell and run at 10 mA of constant current for a slab gel 0 75 mm thick until the bromphenol blue tracking dye enters the separating gel Then increase the current to 15 mA For a standard 16 cm gel sandwich 4 mA per 0 75 mm thick gel will run 15 hr i e overnight 15 mA per 0 75 mm gel will take 4 to 5 hr To run two gels or a 1 5 mm thick gel simply double the current When running a 1 5 mm gel at 30 mA the temperature must be controlled 10 to 20 C with a circulating constant temperature water bath to prevent smiling curvature in the migratory band Temperatures lt 5 C should not be used because SDS in the running buffer will precipitate If the level of
86. wells with native protein standards Add an equal volume of electrophoresis buffer to any empty wells to prevent spreading of adjoining lanes Mobility R markers require special consideration in nondenaturing gel systems For cationic systems cytochrome c pI 9 to 10 5 to 10 ug lane works well as an R marker Bromphenol blue 10 ug ml is a suitable marker for anionic systems The marker should be included in the solubilization buffer with the sample Perform the separation 9 Assemble the gel unit fill the upper and lower buffer chambers with electrophoresis buffer and connect the unit to the power supply Set current to 30 mA for a 1 5 mm thick gel 15 mA for a 0 75 mm thick gel If the protein is negatively charged under the separation conditions then the standard SDS PAGE electrode polarity should be used proteins will migrate to the anode or positive electrode see UNIT 6 1 If the protein is positively charged then the electrodes should be reversed at the power supply i e red high voltage cable to the black output and black high voltage lead to the red output so the positively charged protein migrates to the negative cathode Current Protocols in Cell Biology Table 6 5 4 Recipes for Glycine Nondenaturing Polyacrylamide Gels pH Range 8 6 to 10 6 Final acrylamide concentration in gel 5 7 5 10 12 5 15 17 5 20 Stock solution 30 acrylamide 0 8 6 7 10 13 3 16 8 20 23 32 26 6 bisacrylamide 4x gl
87. with the primary antibody the membrane is washed and the antibody antigen complexes are identified with horseradish peroxidase HRPO or alkaline phosphatase enzymes coupled to the secondary anti IgG antibody e g goat anti rabbit IgG The enzymes are attached directly see Basic Protocol 2 or via an avidin biotin bridge see Alternate Protocol 3 to the secondary antibody Chromogenic or luminescent substrates see Basic Protocol 3 and Alternate Protocol 4 are then used to visualize the activity Finally membranes may be stripped and reprobed see Support Protocol 3 NOTE When handling gels and membranes wear powder free gloves PROTEIN BLOTTING WITH TANK TRANSFER SYSTEMS In this procedure blotting is performed in a tank of buffer with the gel in a vertical orientation completely submerged between two large electrode panels In some systems up to four gels can be transferred at one time For difficult to transfer proteins gt 100 kDa or hydrophobic e g myosin tank blotting is preferable to semidry systems see Basic Protocol 2 because prolonged transfers are possible without buffer depletion However transfers gt 1 hr at high power require cooling using a heat exchanger and a circulating water bath that can maintain a constant transfer temperature of 10 to 20 C Materials Samples for analysis Protein molecular weight standards UNIT 6 1 prestained Sigma or Bio Rad or biotinylated Vector Labs or Sigma Transfer buff
88. 0 4354 Key References Bjerrum O J and Schafer Nielsen C 1986 Buffer systems and transfer parameters for semidry electroblotting with a horizontal apparatus Jn Electrophoresis 86 M J Dunn ed pp 315 327 VCH Publishers Deerfield Beach Fla Describes the semidry blotting system Gillespie and Hudspeth 1991 See above Describes alkaline phosphatase luminescent detec tion methods Harlow and Lane 1988 See above Details alternative detection methods Salinovich O and Montelaro R C 1986 Revers ible staining and peptide mapping of proteins transferred to nitrocellulose after separation by sodium dodecyl sulfate polyacrylamide gel elec trophoresis Anal Biochem 156 341 347 Describes the use of Ponceau S staining for im munoblotting Schneppenheim et al 1991 See above Details peroxidase based luminescent detection methods Contributed by Sean R Gallagher Motorola Corporation Tempe Arizona Scott E Winston and Steven A Fuller tank transfer systems Univax Biologics Rockville Maryland John G R Hurrell tank transfer systems reversible staining of proteins Boehringer Mannheim Biochemicals Indianapolis Indiana Current Protocols in Cell Biology Detection and Quantitation of Radiolabeled Proteins in Gels and Blots This unit presents procedures for visualizing and quantitating radiolabeled proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis
89. 0 staining solution see recipe 10 v v acetic acid a hores 50 ml Erlenmeyer side arm flasks are yee Immunoblotting 6 1 11 Current Protocols in Cell Biology Supplement 37 ALTERNATE PROTOCOL 2 One Dimensional SDS PAGE 6 1 12 Supplement 37 Table 6 1 6 Molecular Weights of Peptide Standards for Polyacry lamide Gel Electrophoresis Peptide Molecular weight Da Myoglobin polypeptide backbone 16 950 Myoglobin 1 131 14 440 Myoglobin 56 153 10 600 Myoglobin 56 131 8 160 Myoglobin 1 55 6 210 Glucagon 3 480 Myoglobin 132 153 2 510 Peptide standards are commercially available from Sigma Aldrich See Sigma Aldrich Technical Bulletin MWSDS70 L for molecular weight markers for proteins 1 Prepare and pour the separating and stacking gels see Basic Protocol 1 steps 1 to 11 using the recipes in Table 6 1 5 2 Prepare the sample see Basic Protocol 1 step 12 but substitute 2x tricine sample buffer for the 2x SDS sample buffer and heat the sample at 40 C for 30 to 60 min to improve solubilization prior to loading Use peptide molecular weight standards Table 6 1 6 If proteolytic activity is a problem see Basic Protocol 1 step 12 heating samples to 100 C for 3 to 5 min may be required 3 Set up the electrophoresis apparatus and load the gel see Basic Protocol 1 steps 13 to 20 but use cathode buffer or water to rinse the wells use cathode buffer in the upper buffer chamber and use a
90. 12 apply here 3 Assemble the electrophoresis apparatus and load the sample see Basic Protocol 1 steps 13 to 20 using the phosphate SDS electrophoresis buffer Load empty wells with 1x phosphate SDS sample buffer 4 Connect the power supply and start the run with 15 mA per 0 75 mm thick gel until the tracking dye has entered the gel Continue electrophoresis at 30 mA for 3 hr 5 gel 5 hr 10 gel 8 hr 15 gel or until the dye reaches the bottom of the gel Use temperature control if available to maintain the gel at 15 to 20 C SDS will precipitate below 15 C in this system 5 Disassemble the gel see Basic Protocol 1 steps 23 to 26 See Safety Considerations in introduction Proteins in the gel may now be stained CASTING AND RUNNING ULTRATHIN GELS Ultrathin gels provide superb resolution but are difficult to handle In this application gels are cast on GelBond a Mylar support material Silver staining is recommended for the best resolution Combs and spacers for gels lt 0 5 mm thick are not readily available for most protein electrophoresis units However by adapting combs and spacers used for DNA sequencing casting gels from 0 2 to 0 5 mm thick is straightforward Additional Materials also see Basic Protocol 1 95 v v ethanol GelBond Lonza cut to a size slightly smaller than the gel plate dimensions Glue stick Ink roller available from art supply stores Combs and spacers 0 19 to 0 5 mm sequencin
91. 3 na 18 37 ng blots NBT BCIP solution long procedure cross reaction with carbonic anhydrase Pro Q Glycoprotein 4 5 1 na not detected can save and re image Detection Kit with 2 na lt 15 6 ng blots Con A alkaline 3 na 15 6 ng can strip and reprobe phosphatase see Basic can post stain with total Protocol 2 protein stains stains specific subsets of glycoproteins long procedure Dansyl hydrazine 4 9 1 1 25 2 5 ng not tested inexpensive 2 1 25 2 5 ng requires longer exposure 3 16 19 ng for competitive brightness low level non specific detection of unglycosylated proteins requires hot acidified DMSO Digoxigenin 3 O 5 6 11 1 na 2 ng good sensitivity succinyl e aminocaproic 2 na 5 9 ng can save and reimage acid hydrazide 3 na 18 37 ng blots Anti digoxigenin alkaline long procedure phosphatase Stain with gt cross reaction with NBT x phosphate carbonic anhydrase Pro Q Emerald 300 Dye 2 blots 7 1 300 pg 2 ng can use either on blots see Basic Protocol 1 4 gels 2 300 pg 18 ng or in gels and Alternate Protocol 3 1 2 ng 9 ng great sensitivity can save and re image blots short procedure can counterstain unglycosylated proteins with SYPRO Ruby dye Abbreviations CHO carbohydrate na not applicable Current Protocols in Cell Biology 6 8 13 Supplement 16 Fluorescence Detection of Glycoproteins in Gels and
92. 7 ml of light low concentration acrylamide gel solution into the reservoir chamber for one 0 75 mm thick gradient gel A practice run with heavy and light solutions is recommended Bromphenol blue should be added to the heavy solution to demonstrate linearity of the practice gradient Open the interconnecting valve briefly to allow a small amount 200 ul of light solution to flow through the valve and into the mixing chamber The presence of air bubbles in the interconnecting valve may obstruct the flow between chambers during casting Add 7 ml of heavy high concentration acrylamide gel solution to the mixing chamber Add 23 ul of 10 ammonium persulfate and 2 3 ul TEMED per 7 ml acrylamide solution to each chamber Mix the solutions in each chamber with a disposable pipet Form the gradient and cast the gel 8 Open the interconnecting valve completely Some of the heavy solution will flow back into the reservoir chamber containing light solution as the two chambers equilibrate This will not affect the formation of the gradient Current Protocols in Cell Biology 9 Turn on the magnetic stirrer and adjust the rate to produce a slight vortex in the mixing chamber 10 Open the outlet of the gradient maker slowly Adjust the outlet valve to a flow rate of 2 ml min If using a peristaltic pump calibrate the flow rate with a graduated cylinder prior to casting the gel Some adjustment of the flow rate may be necessar
93. CHAPTER 6 Electrophoresis and Immunoblotting INTRODUCTION he development of powerful new technologies is a major driving force of scientific progress A good example of this is the role that electrophoretic techniques have played in the evolution of modern cell biology Electrophoresis and related applications have contributed greatly to the understanding of the molecular bases of cell structure and function The combination of high resolution ease of use speed low cost and versatility of electro phoretic techniques is unmatched by any other method used to separate proteins It is for this reason that electrophoresis is an indispensable tool in any cell biology laboratory and that papers describing basic techniques of protein electrophoresis e g Laemmli 1970 and O Farrell 1975 to name just a couple are among the most cited articles in this field Laemmli s technique of discontinuous gel electrophoresis in the presence of SDS for example continues to be widely used and referenced almost 30 years after publication Thus no book of techniques in cell biology would be complete without a detailed description of electrophoretic techniques Chapter 6 begins with unrr6 1 which is a collection of state of the art protocols for analyzing proteins by one dimensional electrophoresis under denaturing conditions on polyacrylamide gels Sodium dodecyl sulfate SDS in combination with a reducing agent and heat is most often used as a denatura
94. DAB 3 3 diaminobenzidine HRPO horseradish peroxidase NBT nitroblue tetrazolium TMB 3 3 5 5 tetramethylbenzidine Recipes and suppliers are listed in Reagents and Solutions except for TMP for which use of a kit is recommended See Commentary for further details 4DAB N iCly can be used without the nickel enhancement but it is much less sensitive McKimm Breschkin 1990 reported that if nitrocellulose filters are first treated with 1 dextran sulfate for 10 min in 10 mM citrate EDTA pH 5 0 TMB precipitates onto the membrane with a sensitivity much greater than 4CN or DAB and equal to or better than that of BCIP NBT JLumi Phos 530 contains dioxetane phosphate MgCly CTAB cetyltrimethylammonium bromide and fluorescent enhancer in a pH 9 6 buffer ALTERNATE PROTOCOL 4 Immunoblotting and Immunodetection 6 2 12 VISUALIZATION WITH LUMINESCENT SUBSTRATES Antigens can also be visualized with luminescent substrates Detection with light offers both speed and enhanced sensitivity over chromogenic and radioisotopic procedures After the final wash the blot is immersed in a substrate solution containing luminol for horseradish peroxidase HRPO systems or dioxetane phosphate for alkaline phosphatase AP systems sealed in thin plastic wrap and placed firmly against film Exposures range from a few seconds to several hours although typically strong signals appear within a few seconds or minutes Current Prot
95. F gel water overlay electrophoresis stacking gel central cooling core unpolymerized polymerized stacking gel outer inner outer inner plate plate plate plate Figure 6 4 2 Casting the second dimension gel and loading the IEF gel A The stacking gel solution should reach to the upper edge of the beveled plate and then the gel solution has to be overlaid with a minimum volume of water The water will stay on the surface because of surface tension B After polymerization the gel is mounted on the central cooling core of the electropho resis unit and the equilibrated IEF gel is placed on top of the polymerized stacking gel Excess buffer is removed and the IEF gel is overlaid with hot agarose equilibration buffer mixture After the agarose solidifies the upper electrophoresis chamber is filled with buffer Cast the second dimension gels 1 Assemble the glass plate sandwich of an electrophoresis apparatus using a beveled plate for the shorter side of the gel sandwich A beveled plate provides more space for a thicker IEF gel and will accommodate a first dimension gel that is at least 1 to 2 mm larger than the thickness of the second dimen sion gel 2 If the thickness of the first dimension gel exceeds that of the second dimension gel pour a separating gel of the desired acrylamide concentration and immediately overlay with water to produce a smooth surface The separating gel height should be a minimum
96. Final concentrations are 6 25 mM Tris Cl 48 mM glycine and 0 025 w v SDS Final pH is 8 3 Store up to 1 week at room temperature Four liters are required for the application Electrophoresis and Immunoblotting 6 7 9 Current Protocols in Cell Biology Supplement 15 Table 6 7 1 Troubleshooting Guide for Agarose Electrophoresis and Immunoblotting Problem Possible cause Solution Agarose electrophoresis Run time too long or too short Band spreads into other lanes Samples leak underneath gel Bands migrate at different rates Voltage too high or too low surrounding gel comb Gel cast unevenly visualized as smiles frowns and sneers Uneven heat distribution Bands not in straight lines visualized Artifacts in wells as a wiggle Bromphenol turns yellow Immunoblotting High background Weak signal or no reaction Insufficient blocking Overdevelopment Protein contamination Incomplete washing Primary or secondary antibody too concentrated Sample load insufficient Low antibody specificity Antigen not transferred Conjugate not active Sample diffusing out of well or into Bottom of well torn when removing pH change during electrophoresis Buffer concentration too high or too low Verify buffer preparation Review and increase or decrease voltage or current settings Minimize time for sample loading and start electrophoresis promp
97. Harbor N Y Harper D R and Murphy G 1991 Nonuniform variation in band pattern with luminol horserad ish peroxidase Western blotting Anal Biochem 192 59 63 Kaufmann S H Ewing C M and Shaper J H 1987 The erasable Western blot Anal Biochem 161 89 95 Klein D Kern R M and Sokol R Z 1995 A method for quantification and correction of pro teins after transfer to immobilization mem branes Biochem Mol Biol Int 36 59 66 Mandrell R E and Zollinger W D 1984 Use of zwitterionic detergent for the restoration of anti body binding capacity of electroblotted menin gococcal outer membrane proteins J Immunol Methods 67 1 11 McKimm Breschkin J L 1990 The use of tetra methylbenzidine for solid phase immunoassays J Immunological Methods 135 277 280 Millipore 1990 Protein blotting protocols for Im mobilon P transfer membrane Bedford Mass Electrophoresis and Immunoblotting 6 2 19 Immunoblotting and Immunodetection 6 2 20 Moos M 1992 Isolation of proteins for microse quence analysis Jn Current Protocols in Immu nology J E Coligan A M Kruisbeek D H Margulies E M Shevach and W Strober eds pp 8 7 1 8 7 12 Greene Publishing Associates and John Wiley amp Sons New York Pampori N A Pampori M K and Shapiro B H 1995 Dilution of the chemiluminescence re agents reduces the background noise on Western blots BioTechniques 18 588 590 Peluso
98. IX 3 Estimate relative mobilities of the proteins An example of a stained gel is shown in Figure 6 5 1 A minimum of four gel concentrations is recommended In Figure 6 5 1 Sigma native molecular weight standards were separated on 5 7 5 10 and 12 5 acrylamide gels 5 1 7 7 10 3 and 12 8 T respectively Plot log R against gel concentration T Fig 6 5 2 Determine the slope of K using linear regression Plot log K of the curves from step 6 against log molecular weight of the standards Fig 6 5 3 Determine the slope using linear regression Estimate the size of the standards and unknowns from the generated curve Ferguson plot Use the curve generated by linear regression to estimate the predicted size of the standard for comparison to the actual size stated by the supplier This indicates the accuracy of the curve The log K lt D value y of the unknown is then used to predict the molecular weight x Current Protocols in Cell Biology lactalbumin carbonic anhydrase Figure 6 5 1 Separation of native protein standards under nondenaturing conditions by discontinuous amp E gt 2 c E 2 2 2 olyacrylamide gel electrophoresis at relative 2 E 3 polyacry g p mobility g 8 D 12 8 T Approximately 20 ug protein a was loaded per lane on a 1 5 mm thick 0 rT 16 cm long gel The gel and samples o lt were prepared according to the 0 1 naan Al
99. Isoelectric focusing using soluble ampholytes Soluble ampholytes are mixtures of low mo lecular weight organic compounds with differing side chain pK values that provide buffering ca pacity In an IEF gel the ampholytes migrate to their isoelectric point where they provide buffer Current Protocols in Cell Biology Table 6 4 2 Size Options for Two Dimensional Gel Electrophoresis First dimension gel Second dimension gel Gel type Diameter D Length L Thickness T Height H Purpose Comments mm cm mm cm Microgels minigels lt 1 5 lt 10 lt D lt 10 Analytical 1 4 lt 1 5 lt 10 gt D lt 10 Analytical 3 6 gt 1 5 lt 10 lt D4 lt 10 Analytical preparative 1 2 7 8 Full size gels lt 1 5 12 18 lt D 12 18 Analytical 1 4 lt 1 5 12 18 gt D 12 18 Analytical 3 6 gt 1 5 12 18 lt D1 12 18 Analytical preparative 1 2 7 8 Giant gels lt 1 5 gt 20 gt D gt 20 Analytical 4 6 9 gt 1 5 gt 20 lt D1 gt 20 Analytical preparative 1 3 7 The second dimension gel width has to be at least equal to the IEF tube gel height Key to comments 1 tube gel cannot be placed directly on top of second dimension gel and use of agarose is recommended 2 use of stacking gel is recommended 3 extrusion and handling are relatively difficult 4 total protein load is limited to usually lt 50 ug for whole cell or tissue extracts 5 tube gel can be placed directly on top of second dimension gel and use of agarose is not ne
100. Kirkegaard amp Perry Moss and Vector Labs Blocking buffer Colorimetric detection For nitrocellulose and PVDF 0 1 v v Tween 20 in TBS TTBS APPENDIX 24 For neutral and positively charged nylon Tris buffered saline TBS APPENDIX 24 containing 10 w v nonfat dry milk Prepare just before use TTBS can be stored lt 1 week at 4 C Luminescence detection For nitrocellulose PVDF and neutral nylon e g Pall Biodyne A 0 2 casein e g Hammarsten grade or I Block Tropix in TTBS APPENDIX 24 Prepare just before use continued Current Protocols in Cell Biology For positively charged nylon 6 w v casein 1 v v polyvinyl pyrrolidone PVP in TTBS APPENDIX 24 With constant mixing add casein and PVP to warm 65 C TTBS Stir for 5 min Cool before use Prepare just before use 4CN visualization solution Mix 20 ml ice cold methanol with 60 mg 4CN Separately mix 60 ul of 30 H O with 100 ml TBS 4PPENDIX24A at room temperature Rapidly mix the two solutions and use immediately DAB NiCl visualization solution 5 ml 100 mM Tris Cl pH 7 5 4PPENDIX 24 100 ul DAB stock 40 mg ml in H O stored in 100 11 aliquots at 20 C 25 ul NiCl stock 80 mg ml in H O stored in 100 1 aliquots at 20 C 15 ul 3 H O Mix just before use CAUTION Handle DAB carefully wearing gloves and mask it is a carcinogen Suppliers of peroxidase substrates are Sigma Kirkegaard amp Perry Moss and Vector Labs Dioxetane p
101. NaOH 1 Wash blot 5 min in distilled water In order to effectively reprobe the membranes casein for AP systems or nonfat dry milk must be used as the blocking agent Chromogenic development leaves a permanent stain on the membrane that is difficult to remove and should not be used when reprobing The stain can interfere with subsequent analysis ifreactive bands from sequential immunostain ings are close together 2 Transfer to 0 2 M NaOH and wash 5 min 3 Wash blot 5 min in distilled water 4 Proceed with immunoprobing procedure see Basic Protocol 2 and Alternate Proto col 3 Casein or nonfat dry milk is recommended as blocking agent when reprobing membranes REAGENTS AND SOLUTIONS Use deionized or distilled water in all recipes and protocol steps For common stock solutions see APPENDIX 24 for suppliers see SUPPLIERS APPENDIX For selection of appropriate chromogenic or luminescent solutions and for definition of abbreviations see Table 6 2 1 Alkaline phosphate substrate buffer 100 mM Tris Cl pH 9 5 100 mM NaCl 5 mM MgCl BCIP NBT visualization solution Mix 33 ul NBT stock 100 mg NBT in 2 ml at 70 DMF stored lt 1 year at 4 C and 5 ml alkaline phosphate substrate buffer see recipe Add 17 ul BCIP stock 100 mg BCIP in 2 ml of 100 DMF stored lt 1 year at 4 C and mix Stable 1 hr at room temperature Recipe is from Harlow and Lane 1988 Alternatively BCIP NBT substrates may be purchased from Sigma
102. O sulfate free radical Figure 6 1 7 Structures of acrylamide and bisacrylamide and the associated reaction producing the polyacrylamide matrix used for protein separation and trailing ions After leaving the stacking gel the protein enters the separating gel The separating gel has a smaller pore size a higher salt concentration and higher pH compared to the stacking gel In the separating gel the glycine ions migrate past the proteins and Current Protocols in Cell Biology the proteins are separated according to either molecular size in a denaturing gel containing SDS or molecular shape size and charge in a nondenaturing gel Proteins are denatured by heating in the presence of a low molecular weight thiol Electrophoresis and Immunoblotting 6 1 33 Supplement 37 One Dimensional SDS PAGE 6 1 34 Supplement 37 ff i HO 2 mercaptoethanol O sodium dodecyl sulfate HO HS dithiothreitol No Na d O Figure 6 1 8 Structures of sodium dodecyl sulfate dithiothreitol and 2 mercaptoethanol used to break disulfide bonds in proteins so they are fully denatured 2 ME or DTT and SDS Fig 6 1 8 Most proteins bind SDS in a constant weight ratio leading to identical charge densities for the de natured proteins Thus the SDS protein com plexes migrate in the polyacrylamide gel ac cording to size not charge Most proteins are resolved on polyacrylamide gels containing f
103. PD CSPD Lumigen PPD Lumi Phos 530 Oxidized products form purple precipitate Forms dark brown precipitate Forms dark purple stain BCIP hydrolysis produces indigo precipitate after oxidation with NBT reduced NBT precipitates dark blue gray stain results Oxidized luminol substrate gives off blue light p iodophenol increases light output Dephosphorylated substrate gives off light Not very sensitive Tween 20 inhibits reaction fades rapidly upon exposure to light More sensitive than 4CN but potentially carcinogenic resulting membrane easily scanned More stable less toxic than DAB NiCl may be somewhat more sensitive can be used with all membrane types kits available from Kirkegaard amp Perry TSI Moss and Vector Labs More sensitive and reliable than other AP precipitating substrates note that phosphate inhibits AP activity Very convenient sensitive system reaction detected within a few seconds to hr Protocol described gives reasonable sensitivity on all membrane types consult instructions of reagent manufacturer for maximum sensi tivity and minimum background see Troubleshooting Abbreviations AMPPD or Lumigen PPD disodium 3 4 methoxyspiro 1 2 dioxetane 3 2 tricyclo 3 3 1 17 gt 7 decan 4 yl pheny phosphate AP alkaline phosphatase BCIP 5 bromo 4 chloro 3 indolyl phosphate 4CN 4 chloro 1 napthol CSPD AMPPD with substituted chlorine moiety on adamantine ring
104. R W and Rosenberg G H 1987 Quantita tive electrotransfer of proteins from sodium do decyl sulfate polyacrylamide gels onto positively charged nylon membranes Anal Biochem 162 389 398 Perides G Plagens U and Traub P 1986 Protein transfer from fixed stained and dried polyacry lamide gels and immunoblot with protein A gold Anal Biochem 152 94 99 Sandhu G S Eckloff B W and Kline B C 1991 Chemiluminescent substrates increase sensitiv ity of antigen detection in Western blots Bio Techniques 11 14 16 Schneppenheim R Budde U Dahlmann N and Rautenberg P 1991 Luminography a new highly sensitive visualization method for electro phoresis Electrophoresis 12 367 372 Suck R W L and Krupinska K 1996 Repeated probing of Western blots obtained from Coomas sie Brilliant Blue stained or unstained polyacry lamide gels BioTechniques 21 418 422 Talbot P V Knobler R L and Buchmeier M 1984 Western and dot immunoblotting analysis of viral antigens and antibodies Application to murine hepatitis virus J Immunol Methods 73 177 188 Tesfaigzi J Smith Harrison W and Carlson D M 1994 A simple method for reusing western blots on PVDF membranes BioTechniques 17 268 269 Towbin H Staehelin T and Gordon J 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets Procedure and some applications Proc Natl Acad Sci U S A 76 435
105. S Conditions for transfer are the same as those used for SDS PAGE gels UNIT 6 2 Before transfer mark the ferritin marker with a pen on the membrane since it is difficult to see afterwards 3 Optional Partially remove the transferred Coomassie blue from the PVDF mem branes by incubation in destaining solution for 30 min Immunodetection can also be performed without the destaining step Coomassie blue cannot be removed completely and shows strong fluorescence at several wavelengths Therefore detection with fluorescently labeled antibodies cannot be accomplished In this case a second dimension SDS PAGE has to be done Basic Protocol 2 4 Block the membrane and immunodetect under standard conditions UNIT 6 2 NATIVE ANTIBODY BASED MOBILITY SHIFT NAMOS ASSAY This protocol is a variant of Basic Protocol 1 and describes a method to determine the stoichiometry of multiprotein complexes based on BN PAGE It makes use of the fact that the proteins and protein complexes are separated in their native state Antibodies that recognize a certain subunit of a protein complex are added to the sample before separation by BN PAGE This allows the formation of super complexes comprising the protein complex and the antibody Since the migration distance in BN PAGE depends on the size of a protein complex it directly reflects the number of bound antibodies and thus the copy number of the subunit they bind to This method is called the Native Ant
106. The solution is also referred to as 1X TAE SDS Two liters are required for the application Fixing buffer 50 ml isopropanol 25 20 ml glacial acetic acid 10 130 ml H O Prepare fresh on the day of use High salt wash buffer 56 78 g Na HPO 0 1 M final 233 6 g NaCl 1 M final Adjust pH to 7 0 with concentrated HC1 Add H O to 4 liters Store up to 3 months at room temperature Current Protocols in Cell Biology Human IgG 2 2 0 g human IgG in 100 ml BSB see recipe Store in aliquots up to 1 year at 20 C In gel sample buffer Stock solution 0 01M Na HPO Adjust pH to 7 0 with HCl Filter and store up to 3 months at room temperature Working solution To 3 ml stock solution add 20 64 mg iodoacetamide 37 5 mg SDS Prepare fresh Sample buffer 2x 20 ml 10x TAE see recipe 1 ml 20 w v SDS APPENDIX 2A 20 ml glycerol 0 2 g bromphenol blue Add H 0 to 200 ml Store up to 1 year at room temperature TAE 10x 48 4 g Tris base 400 mM 20 ml 0 5M EDTA 10 mM APPENDIX 2A Adjust pH to 7 8 with glacial acetic acid Adjust volume to 1 liter Store up to 1 year at room temperature Transfer buffer without methanol 0 25x and 10x For a 10X solution 250 mM Tris Cl pH 8 3 APPENDIX 24 1 92 M glycine 1 0 w v SDS Store up to 1 year at room temperature This is the stock solution for transfer buffer and is also known as Tris glycine SDS TG SDS For a 0 25X solution Dilute 10x transfer buffer 1 40 in distilled water
107. This protocol describes the general procedure of casting custom made Immobiline gels The Reswelling Cassette used in Basic Protocol 2 for rehydrating gels is employed for casting the gels which are polyacrylamide gels poured with a gradient of Immobilines following instructions provided by Amersham Pharmacia Biotech application note 324 Additional Materials also see Support Protocol 2 GelBond PAG film Amersham Pharmacia Biotech Immobiline solutions Amersham Pharmacia Biotech 2 5 v v glycerol Gradient maker Orbital shaker Additional materials and equipment for rehydrating immobilized pH gradient gels see Basic Protocol 2 Cast the gel 1 Coat the plate with the U frame with Repel Silane to prevent the gel from sticking to the glass plate 2 Place a glass plate on a clean flat surface and wet with several drops of water Cover with a sheet of GelBond PAG film hydrophilic side up Use the roller to remove any air bubbles trapped between the film and the glass plate 3 Place the plate with the U frame on top of the GelBond PAG film Clamp the plates together on three sides Current Protocols in Cell Biology 4 Mix the Immobiline solutions following the instructions provided by Amersham Pharmacia Biotech application note 324 to prepare the desired pH range Cast the pH gradient gel using a gradient maker Once the catalysts have been added to the gel solution it is important to work quickly to ensure that the gradient
108. a darkroom illuminated with a safelight place the sample e g dried gel or filter in the film cassette Cover the sample with plastic wrap to prevent it from sticking to the film and contaminating the cassette with radioactivity The safelight should be a bulb of lt 15 W that is equipped with a Kodak GBX 2 red filter or equivalent Fluorescent glow in the dark ink available at craft stores is a convenient way to mark samples exposed to film The ink can be spread on adhesive labels which in turn are placed on the plastic wrap around the edge of the sample If exposed to light prior to autoradiog raphy the ink will fluoresce and expose the film making it possible to orient the film image on the dried gel after developing 2 Place asheet of X ray film on top of the sample then close and secure the film cassette see Fig 6 3 1 If preflashed film is used for direct exposure see Support Protocol 3 the exposed side should face the sample Preflashed film should be used if sample is weakly radioactive or if quantitation of the radioactivity is desired For single coated film the emulsion layer should face the sample If a paper cassette is used particle board supports the same size as the cassette should be placed on either side and secured with the metal binder clips This will ensure that the sample and film are held in contact and do not shift during exposure Current Protocols in Cell Biology film cassette inte
109. acers or fewer gels calculate volumes using the equation in the annotation to step 4 The recipes are based on the SDS denaturing discontinuous buffer system of Laemmli 1970 Pall reagents and solutions used in the protocol must be prepared with Milli Q purified water or equivalent Volumes in table body are in milliliters The desired percentage of acrylamide in separating gel depends on the molecular size of the protein being separated See Basic Protocol 1 annotation to step 3 4Best to prepare fresh Failure to form a firm gel usually indicates a problem with the persulfate TEMED or both 2 Assemble glass sandwiches and stack them in the casting chamber Stack up to ten 1 5 mm gels and fill in extra space with acrylic blocks or polycarbonate sheets to hold the sandwiches tightly in place Make sure the spacers are straight along the top right and left edges of the glass plates and that all edges of the stack are flush The presence of loosely fitting sandwiches in the caster will lead to unevenly cast gels creating distortions during electrophoresis Polycarbonate inhibits gel polymerization Therefore if polycarbonate sheets are placed in the caster before and after the set of glass sandwiches the entire set will slide out as one block after polymerization Placing polycarbonate sheets between each gel sandwich makes them easier to separate from one another after polymerization 3 Place the front sealing plate on the castin
110. ach in preliminary experiments including detergents that allow successful co immunopurifications of the proteins expected to be present in a common complex Current Protocols in Cell Biology In the BN lysis buffer orthovanadate is in cluded to inhibit phosphatases Since ortho vanadate is a small molecule it is rapidly separated from the sample during the BN PAGE run Since it is a reversible inhibitor phosphatases become active once orthovana date is removed Thus phosphorylation of the proteins might be lost during the native electrophoresis where active phosphatases might be present Since pervanadate irre versibly inhibits phosphatases it should not be omitted from the BN dialysis buffer when phosphorylation of proteins needs to be preserved One common surprise that is encountered in performing BN PAGE is that the size of the protein complex of interest does not match the expected value First the detergent micelle around transmembrane regions adds to the size of the corresponding protein Hence the size of the protein or protein complex might be larger than that obtained by simply adding the molecular weights of the individual sub units Thus to estimate the size of transmem brane proteins complexes the mass calibra tion markers should also be transmembrane proteins solubilized with the same detergent Some water soluble proteins match the cali bration curve of dodecylmaltoside solubilized transmembrane prote
111. activity also load the highest concentration of the monoclonal antibody alone i e without adding the protein protein mixture of interest Electrophoresis and Immunoblotting 6 10 13 Current Protocols in Cell Biology Supplement 38 Two Dimensional Blue Native Polyacrylamide Gel Electrophoresis 6 10 14 Supplement 38 REAGENTS AND SOLUTIONS Use deionized distilled water in all recipes and protocol steps For common stock solutions see APPENDIX 24 for suppliers see SUPPLIERS APPENDIX Acrylamide bisacrylamide mix 50 ml of 19 1 acrylamide bisacrylamide solution 239 ml of 37 5 1 acrylamide bisacrylamide solution Store at room temperature stable at least 1 year This will result in a ratio of 32 1 with 40 acrylamide BN anode buffer Dilute 50 ml of 1 M bis Tris stock solution pH adjusted to 7 0 with HCI store up to 1 year at room temperature to 50 mM final by adding water to 1 liter BN cathode buffer 15 ml 1 M bis Tris stock solution pH adjusted to 7 0 with HCl 15 mM final 50 ml 1 M tricine 50 mM final 0 2 g Coomassie blue G250 not other Coomassie blues 0 02 w v final H20 to 1 liter Store at room temperature stable at least 1 year BN cathode buffer low Coomassie 15 ml 1 M bis Tris stock solution pH adjusted to 7 0 with HCl 15 mM final 50 ml 1 M tricine 50 mM final 0 02 g Coomassie blue G250 not other Coomassie blues 0 002 w v final H20 to 1 liter Store at room temper
112. activity or another criterion 7 Microcentrifuge the cells 15 min at maximum speed 4 C 8 Remove and discard the supernatant using a pipettor or Pasteur pipet Weigh the vial containing the cell pellet Record the wet weight of the cell pellet in mg 9 Freeze the cell pellet in a dry ice ethanol mixture optional Frozen cells can be stored at 80 C for at least several months Prepare the cell pellets for isoelectric focusing 10 Retrieve cell pellets from 80 C storage if samples were frozen 11 Add 400 ul Tris SDS buffer per 50 to 100 mg cell pellet wet weight Keep the cells on ice at all times The total amount of protein in the pellet is roughly 5 of the wet pellet weight Two Dimensional Gel Electrophoresis 6 4 18 Supplement 4 Current Protocols in Cell Biology 12 13 14 15 16 17 Sonicate the sample three times for 3 sec using a sonicator with a microtip at medium power Keep the samples on ice during sonication Use pulse sonication or wait at least 5 min between sonications Minimizing heat genera tion is essential because substantial proteolysis can occur if the sample warms appreciably If additional sonication is necessary i e if the sample is not homogeneous let the sample cool down on ice before the next series of sonications Run a BCA protein assay to determine the protein concentration if sample will be loaded on that basis For maximum accuracy use the same a
113. adient slab gels run under reducing or nonreducing con ditions The only exception is that loading of the first dimension BN gel strip requires a broad flat well The BN gel strip has to be fit between the glass plates of the second gel The easiest way is to slightly increase the thickness of the second dimension compared to the first dimension If this is done by putting cellophane tape on the spacers of the second dimension gel the BN gel strip can be inserted without breaking and still sit firmly enough to prevent any movement during the electrophoresis Alternatively procedures as described in UNIT 6 4 can be employed This protocol describes all the specific steps required for successfully casting and running the second dimension SDS PAGE with extensive reference to UNIT 6 1 Typical expected results are depicted in Figure 6 10 1 Materials 2x SDS sample buffer UNIT 6 1 1x SDS electrophoresis buffer UNIT 6 1 Lane from a BN PAGE gel Basic Protocol 1 Thin adhesive tape e g tesa tape http www tesatape com Platform shaker Additional reagents and equipment for SDS PAGE uniT 6 1 and two dimensional gel electrophoresis UNIT 6 4 Cast the second dimension SDS gels 1 Take spacers of the same thickness as the ones that were used for the first dimension see Basic Protocol 1 Wrap thin adhesive tape once around them in order to increase their thickness slightly 2 Assemble the glass plate sandwich using these spacers
114. age acquisition was adjusted to increase the contrast of the displayed image Although useful for images with a narrow range of informative intensity values increasing the contrast can lead to a loss of low and high values C Decreasing the brightness reduces peak values but also leads to a loss of the weak bands and original background D Gamma adjusts raw data to appear more visually accurate Note that this leads to a loss of fidelity between the adjusted image and the original E Saturation indicates that the detector is reporting its maximum value or that the dynamic range for the visualization method has been exceeded Current Protocols in Cell Biology Electrophoresis and Immunoblotting 6 9 3 Supplement 16 Digital Electrophoresis Analysis 6 9 4 Supplement 16 high resolution peaks medium resolution peaks Intensity 42um 168 um 840 um Pixel position Figure 6 9 2 The effect of spatial resolution on the ability to detect closely spaced objects Whole cell protein lysates from E coli were separated using SDS PAGE and visualized with Coomassie blue staining An image was captured at 42 um 600 dpi 168 um 150 dpi and 840 um 30 dpi from a segment of the lane and a lane profile was generated for each image The lane profiles have offset intensities to allow for comparison Only major bands can be detected with the low resolution image 840 um at higher r
115. age of acrylamide used for the second Electrophoresis and Immunoblotting 6 4 35 Supplement 4 Two Dimensional Gel Electrophoresis 6 4 36 Supplement 4 dimension gel A complex protein mixture such as a whole cell extract should produce more than 1000 silver stained spots distributed over most of the gel area Fewer spots will be de tected with less sensitive detection techniques such as Coomassie blue staining On the other hand separation of radiolabeled proteins and use of multiple exposures permit detection of many low abundance proteins Time Considerations Time requirements are very dependent on gel size and whether an external cooling unit is used to permit faster separations Isoelectric focusing using the standard size gel format described in Basic Protocol 1 with 3 mm tubes is most con veniently done in an overnight run of 16 to 18 hr This separation time can be decreased to 5 to 6 hr using higher voltages and an external cooling device Isoelectric focusing of 18 cm long IPG gels requires 16 to 18 hr Extruding a set of sixteen IEF tube gels and freezing them in equili bration buffer takes 1 to 2 hr including setup time Preparing and running SDS gels is described in UNIT 6 1 It takes 30 min to thaw equilibrate and load two second dimension gels Overall if standard size gels are used without external cooling it will take 3 working days before the results of two dimensional electropho
116. ajority of applications Narrow strips of precast IEF gels immobiline DryStrips or Ready Strip IPG strips may be used to achieve a first dimen sion separation for two dimensional gel electrophoresis and broader precast slab gels Immobiline DryPlates can be used to compare multiple samples after IEF separation only see Support Protocol 2 and Table 6 4 3 In this protocol precast Immobiline DryStrips from Amersham Pharmacia Biotech are rehydrated overnight using the reswelling cassette one to twelve sample strips may be handled at a time samples are applied using sample cup holders and gel strips are isoelectric focused overnight This procedure has been adapted from instruction booklets provided by Amersham Pharmacia Biotech with Immobiline Dry Strip Kits and with the Immobiline DryStrip reswelling tray Recently both Amersham Pharmacia Biotech and Bio Rad have developed newer IEF systems the IPGphor Isoelectric Focusing System and the Protean IEF Cell respec tively These systems simplify IPG strip handling and the overall isoelectric focusing procedure The IPGphor system also integrates IPG strip reswelling and electrophoresis steps in a single strip holder These systems include very high voltage power supplies integrated with efficient cooling units to permit more rapid isoelectric focusing In general premade commercial IPG strips from any supplier can be used with any isoelectric focusing device that can physically accommodate the strip
117. ally de termined by applying the sample to different positions on the gel and estimating the time for the migration patterns to coincide Some proteins may require longer run times to reach their pl Because there is no gradient drift the potential problems with longer run times are limited to sample modifications or drying out of the gel These problems usually can be minimized by including a reducing agent in the rehydration solution and coating the top surface of the gel with paraffin oil The quality of the first dimensional separation is strongly dependent on the purity of the reagents used especially the urea and ampholytes One fairly commonly encountered frustration when soluble ampholyte gels are used is that different batches of ampholytes from the same supplier will sometimes produce markedly different two dimensional gel patterns Therefore it is advisable to purchase an adequate supply of a single lot of ampholytes to meet anticipated needs for an entire study where such an ap proach is feasible However whereas ampho lytes usually have a reasonably long shelf life at 4 C usually up to a year shelf life as well as total ampholyte requirements often cannot be predicted with much certainty When any doubt arises about the purity or quality of ampholytes or any other reagent the reagent should be replaced immediately Constant monitoring of the system performance especially when changing lots of ampholytes urea or
118. alternative staining method e g Basic Protocol 2 or Basic Protocol 4 Rinse the gel further with water i e repeat steps 4 and 5 Place the gel directly onto the surface of the UV transillumination unit Remove the gel from the polyester surface before visualization Allow the SDS front to migrate out of the gel Handle the gel gently wearing gloves that have been rinsed with water Table 6 6 4 Troubleshooting Guide for Staining with Zinc Basic Protocol 4 Problem Cause Solution Protein bands are absent or too faint Blackground does not turn opaque Insufficient contrast due to the use of clear surfaces for visualization Gel is overdeveloped Amount of protein s below detection limit Stain solution or developer are too old Visualize the bands by placing the gel against a dark surface Destain the gel steps 9 and 10 and stain again monitoring the appearance of bands against a dark surface Check protein concentration in original sample Stain the gel with silver Basic Protocol 2 Use fresh stain and developer solutions do not reuse them 6 6 12 Supplement 6 Current Protocols in Cell Biology longer linear ranges than the other staining methods Steinberg et al 1996 Nevertheless in order to check the validity of the measure ments it is always advisable to load in the gel together with the sample s to be quantitated internal controls or a calibration curve w
119. an the low percentage gel because of its high glycerol content This density difference aids in establishment of a uniform gradient inside the glass plates The same gel equipment that is used for normal SDS PAGE can be used but one has to be certain that no traces of SDS are present To ensure absence of any detergent the BN equipment should not be used for SDS PAGE BN gels are poured at room temperature and are cooled to 4 C before samples are loaded in the cold room Alternatively gel apparatuses that allow cooling can be used Precast BN gels have recently been made commercially available but in the author s experience these are not as good as self made gels It is strongly recommended to use multicasting equipment to pour several gels at once This avoids steps of the gradient saves time and ensures best reproducibility for critical comparisons of multiple samples Casting of multiple gradient gels is described in UNIT6 Support Protocols 2 and 3 and Figures 6 1 3 and 6 1 4 Using these protocols BN gels can be prepared using BN specific solutions Materials Low percentage BN separating gel solution see recipe High percentage BN separating gel solution see recipe Isobutyl alcohol 3x BN gel buffer see recipe BN stacking gel solution see recipe 100x pervanadate solution optional if phosphorylation must be preserved see recipe Sample dialyzed cell lysate Support Protocol tissue homogenate purified protein com
120. ansferred to a membrane for immunoblotting UNIT 6 2 If the proteins are radiolabeled they can be detected by autoradiography UNIT 6 3 ELECTROPHORESIS IN TRIS TRICINE BUFFER SYSTEMS Separation of peptides and proteins under 10 to 15 kDa is not possible in the traditional Laemmli discontinuous gel system see Basic Protocol 1 This is due to the comigration of SDS and smaller proteins obscuring the resolution Two approaches to obtain the separation of small proteins and peptides in the range of 5 to 20 kDa are presented the following Tris tricine method and a system using increased buffer concentrations see Alternate Protocol 2 The Tris tricine system uses a modified buffer to separate the SDS and peptides thus improving resolution Several precast gels are available for use with the tricine formulations Table 6 1 4 Current Protocols in Cell Biology Table 6 1 4 Precast Gels Available from Selected Suppliers Format Application Instrument Compatibility Large Mini 2 D Native Peptide SDS Bio Rad Cambrex Hoefer Invitrogen Bio Rad X X X X X X X Cambrex X X X X X X X X X Jule X X X X X X X X X X Invitrogen X X X X X X X X X Two dimensional analysis Table 6 1 5 Recipes for Tricine Peptide Separating and Stacking Gels SEPARATING AND STACKING GELS Stock solution Separating gel Stacking gel 30 w v acrylamide 0 8 w v 9 80 ml 1 62 ml bisacrylamide Tris Cl SDS pH 8 45 10 00 ml 3 10 m
121. arating gel solution with the desired percentage acrylamide Table 6 1 1 omit the stacking gel for the first dimension Stacking gels can usually be avoided in the first dimension by keeping sample volumes small i e lt 10 ul Less than 200 ul of gel solution is required to cast a single 1 2 mm tube gel 12 cm in length Adjust the amounts from Table 6 1 1 accordingly 3 Cast the first dimension polyacrylamide gels in 1 2 mm tubes using a syringe with a long needle see Basic Protocol 1 step 5a Overlay with water and allow the gels to polymerize 4 Prepare samples in 1x SDS sample buffer without any reducing reagents i e no 2 mercaptoethanol or DTT Load the samples and electrophorese until the tracking dye is 1 cm from the bottom of the tube Reduce sample and run the second dimension gel 5 Extrude the gel from the tube see Basic Protocol 1 steps 27 to 29 6 Place the extruded gel in a test tube containing 5 ml reducing buffer Equilibrate 15 min at 37 C with gentle agitation 7 Cast the second dimension separating and stacking gels see Basic Protocol 3 steps 1 to 3 making sure that the top of the stacking gel is at least 5 mm below the top of Current Protocols in Cell Biology ALTERNATE PROTOCOL 3 Electrophoresis and Immunoblotting 6 4 25 Supplement 4 SUPPORT PROTOCOL 7 Two Dimensional Gel Electrophoresis 6 4 26 Supplement 4 the short glass plate Layer water across the e
122. aration under nonreducing conditions in the first dimension followed by reduction of disulfide bonds and separation under reducing conditions in the second dimension Most proteins will migrate equal distances in both dimensions forming a diagonal pattern Proteins containing interchain disulfide bonds will be dissociated into individual subunits and can be resolved in the second di mension gel The approach is similar to that described for two dimensional gel electrophoresis see Basic Protocol 3 except in this protocol the first dimension gels are nonreducing i e 2 mercaptoethanol or dithiothreitol is omitted from sample buffer SDS denaturing gels instead of isoelectric focusing gels Use of 1 2 mm tube gels for the first dimension separation and 1 5 mm slab gels for the second dimension run is recommended Additional Materials also see Basic Protocol 3 Separating and stacking gel solutions see Table 6 1 1 1x SDS sample buffer without reducing agents UNIT 6 1 Reducing buffer see recipe 1 5 w v agarose in reducing buffer see recipe optional for securing first dimension gel on second dimension gel Two dimensional comb optional Additional reagents and equipment for casting tube gels see Basic Protocol 1 SDS PAGE unir 6 1 and protein staining APPENDIX 3 Pour and run the first dimension gel 1 Clean and dry 1 2 mm glass gel tubes for the first dimension gel see Basic Proto col 1 step 1 2 Prepare a sep
123. are highly sensitive and it is critical that all glassware and staining dishes be scrupulously clean Gels and blots should never be touched or otherwise manipulated using bare hands Always wear powder free latex gloves when handling gels and blots during all staining pro cedures for the fluorescence detection of gly coproteins Troubleshooting Should the detection sensitivity obtained using the cited fluorescence methods be subop timal there are two potential sources for the problem either instrumental or chemical With respect to the imaging instrument it is impor tant to clean the surface of the transilluminator after each use with deionized water and a soft cloth e g cheesecloth Otherwise fluores cent dyes can accumulate on the glass surface and cause a high background fluorescence The polyester backing on some pre cast gels is highly fluorescent For maximum sensitivity using a UV transilluminator the gel should be placed polyacrylamide side down and an emis sion filter used to screen out the blue fluores cence of the plastic For UV detection of fluoro phores a 300 nm UV B transilluminator with six 15 watt bulbs is recommended Excitation with different UV light sources such as a sim ple hand held UV lamp will not provide the same level of detection sensitivity as a full fledged transilluminator For all three proce dures described in this unit using a Polaroid camera and Polaroid 667 black and white print
124. are not susceptible to deglycosylation with specific enzymes may readily be identified as glycoproteins After detecting glycoproteins with Pro Q Emerald 300 dye total protein profiles may be detected using SYPRO Ruby protein gel stain SYPRO Ruby protein gel stain interacts noncovalently with basic amino acid residues in proteins The stain is capable of detecting lt 1 ng of protein band making it at least as sensitive as the best silver staining procedures available The orange red fluorescent signal from SYPRO Ruby protein gel stain can be visualized using a standard 300 nm UV UV B illumination source or alternatively may be excited using 470 to 488 nm laser gas discharge or xenon arc sources Materials Protein sample of interest Fix solution see recipe Wash solution see recipe Pro Q Emerald 300 Glycoprotein Gel Stain Kit Molecular Probes containing 50x Pro Q Emerald 300 reagent concentrate in DMF Pro Q Emerald 300 dilution buffer Periodic acid oxidizing reagent see recipe CandyCane glycoprotein molecular weight standards see recipe sufficient volume for approximately 20 gel lanes SYPRO Ruby protein gel stain Deionized high quality water 10 v v methanol or ethanol spectroscopy grade optional 7 v v glacial acetic acid optional Polystyrene staining dishes e g a weighing boat for minigels or larger container for larger gels Orbital shaker UV transilluminator Photographic camera or CCD camera and
125. are rather uniform A drawback of this and other silver staining methods is that they can also stain other macromolecules namely DNA RNA and bacterial lipopolysac charides Procedures for the fluorescent staining of proteins in polyacrylamide gels include cova lent modification with fluorescamine or fluo rescein isothiocyanate and noncovalent stain ing with the hydrophobic probes 1 anili nonaphthalene 8 sulfonic acid or nile red see Current Protocols in Cell Biology Steinberg et al 1996 and references therein A family of fluorescent protein stain reagents including SYPRO Orange SYPRO Red and SYPRO Ruby has recently been introduced and reported to be highly sensitive and protein specific Steinberg et al 1996 These reagents detect protein SDS complexes rather than pro tein functional groups and are compatible with most downstream applications Known inter ferences are colored stains or prosthetic groups Triton X 100 and the excess of SDS at the migration front A simple protocol for protein staining with SYPRO Ruby which is commer cially available and said to be the most sensitive dye within the SYPRO family is described in Basic Protocol 3 A different group of protein staining meth ods for SDS PAGE are based on the reversible formation of SDS precipitates thus allowing visualization of protein bands as clear areas on an opaque background Although SDS precipi tation can be simply achieved by lowering th
126. are removed by pretreat ing the casein with avidin agarose Sigma Anticipated Results Immunoblotting should result in the detec tion of one or more bands Although antibodies directed against a single protein should produce a single band degradation of the sample e g via endogenous proteolytic activity may cause visualization of multiple bands of slightly dif ferent size Multimers will also form spontane ously causing higher molecular weight bands on the blot If simultaneously testing multiple antibodies directed against a complex protein mixture e g using patient sera against SDS PAGE separated viral proteins in AIDS west ern blot test multiple bands will be visualized Typically picogram to nanogram sensitivities are common in protein blotting and immunode tection procedures Time Considerations The entire immunoblotting procedure can be completed in 1 to 2 days depending on transfer time and type of gel Gel electrophore sis requires 4 to 6 hr on a regular gel and 1 hr on a minigel Transfer time can be 1 hr high power transfer to overnight Blocking conju gate incubation and washing each take 30 min to 1 hr Finally substrate incubation requires 10 to 30 min chromogen and a few seconds to several hours luminescence Current Protocols in Cell Biology Literature Cited Bjerrum O J Larsen K P and Heegaard N H H 1988 Nonspecific binding and artifacts speci ficity problems and troubleshoo
127. aster and easier to quantify radioactive samples To enhance radioactive signals solid state scintillation is frequently employed to convert the energy released by radioactive molecules to visible light This is accomplished in several different ways In fluorography see Alternate Protocol 1 organic scintillants are incorporated into the sample to increase the proportion of emitted energy detected from low energy particles e g from H C and S Another method uses high density fluorescent intensifying screens see Support Protocol 2 which are placed next to the sample and used to capture the excess energy of y rays e g those produced by I and high energy particles e g from P CAUTION When working with radioactivity take appropriate precautions to avoid contamination of the experimenter and the surroundings Carry out the experiment and dispose of wastes in appropriately designated areas following the guidelines provided by your local radiation safety officer also see APPENDIX 1D AUTORADIOGRAPHY Autoradiography uses X ray film to visualize and quantitate radioactive molecules that have been electrophoresed through agarose or polyacrylamide gels UNIT 6 1 hybridized to filters e g immunoblots UNIT 6 2 or chromatographed through paper or thin layer plates A photon of light or the B particles and yrays released from radioactive molecules activate silver bromide crystals on the film emulsion
128. ater to 2 liters Make fresh daily Must be degassed prior to use PBS 10x 152 g NaCl 24 g monobasic sodium phosphate anhydrous 1600 ml H O Adjust pH to 6 7 with NaOH Add H O to 2 liters Store at room temperature stable at least 1 month The 1x solution should be pH 7 3 to 7 5 Final concentrations are 130 mM NaCl and 10 mM sodium phosphate PBS with proteolysis inhibitors PBS I buffer 20 ml 10x PBS see recipe Two Dimensional 20 ml 2 w v EDTA pH 7 0 see recipe Gel Electrophoresis continued 6 4 28 Supplement 4 Current Protocols in Cell Biology 200 ul 0 15 M phenylmethylsulfonyl fluoride PMSF in 2 propanol 100 ul 2 mg ml leupeptin see recipe 200 ul 1 mg ml pepstatin see recipe Adjust to pH 7 2 with HCl Add H O to 200 ml Prepare immediately before use Diisopropyl fluorophosphate DFP is a better serine protease inhibitor than PMSF at lower temperatures 0 to 4 C however although both compounds are toxic exceptional caution must be exercised with DFP owing to its volatility If DFP is used work in a chemical fume hood and carefully follow the supplier s precautions DFP and PMSF have half lives on the order of hours in aqueous neutral solutions and the degradation rate increases rapidly as the pH is increased above neutral Make aqueous solutions immediately before use Use of 1 M NaOH is convenient to inactivate residual DFP or PMSF Final concentrations are 10 mM sodium phosphate 130 mM NaC
129. ation related to a specific spot can also be added to the investigator built database including the pI molecular weight amino acid composition sequence and or identity of the protein and any other important attributes correlated with the indicated spot A number of research groups including those of Garrels and Celis Garrels 1989 Garrels and Franza 1989 Celis et al 1991 have extensively charac terized hundreds of spots from specific cell lines and have used multiple methods to characterize proteins of interest The most definitive methods for establishing the identi Current Protocols in Cell Biology ties for proteins of interest detected by computer assisted comparisons are protein sequence analysis and more recently mass spectrometry of tryptic fragments Both methods are compatible with the quantities of protein that can be recovered from two dimensional gels REAGENTS AND SOLUTIONS Use Milli O purified water or equivalent in all recipes and protocol steps For common stock solutions see APPENDIX 24 for suppliers see SUPPLIERS APPENDIX 30 acrylamide 0 8 bisacrylamide 30 g acrylamide 0 8 g bisacrylamide H O to 100 ml Filter solution through 0 2 to 0 45 um filter e g Micro Filtration Systems cellulose nitrate 0 2 um Store at 4 C stable at least 3 months CAUTION Acrylamide is a neurotoxin Wear gloves and a dust mask when handling solid acrylamide Wear gloves when working with acrylamide solution Never pip
130. atterns nor can it deal with large numbers of bands or spots Accuracy of meas urement is a primary reason for using digital analysis on electrophoretically separated pro teins and nucleic acids Two categories of ac curacy are key to digital analysis positional accuracy which is important for mobility de terminations such as molecular weight and quantitative accuracy Positional accuracy is based on both resolu tion of the recording medium and measuring accuracy Silver halide based recording has a theoretical resolution based on 2000 imaging elements silver grains per inch Measurement traditionally occurs using a ruler with an accu racy of 20 to 40 elements 50 to 100 elements per inch In comparison typical digital sys tems have 200 to 600 picture elements pixels per inch The advantage that digital systems have is in measuring accuracy which can occur at the level of a single imaging element Quantitative accuracy is also an issue The amount of material represented by a band or spot is difficult to determine accurately from an image of a gel unless it is a digital image On a digital image the amount present is directly correlated with the derived volume of the band or spot the volume is calculated using the intensity values of the pixels within the object Reproducibility Any technique or measurement is only as good as its ability to be faithfully replicated With software defined routines measurements
131. ature stable at least 1 year BN dialysis buffer Mix 2x BN lysis buffer stock solution see recipe and the detergent stock solution see recipe of choice so that the BN lysis buffer has a final concentration of 1 x and the detergent solution has a final concentration of 0 1 but for digitonin use 0 3 x due to its low critical micelle concentration Add PMSF and sodium orthovanadate see recipe for protease and phosphatase inhibitor stock solutions to a final concentration of 1 x Prepare fresh just before use and cool to 4 C Upon addition of orthovanadate the buffer will assume a yellow color BN gel buffer 3x 150 ml 1 M bis Tris stock solution pH adjusted to 7 0 with HCl 150 mM final 200 ml 1 M 6 aminohexanoic acid Sigma Aldrich stock solution 200 mM final H20 to 1 liter Store at room temperature stable at least 1 year BN lysis buffer Mix 2x BN lysis buffer stock solution see recipe and the detergent stock solution see recipe of choice so that both have a final concentration of 1x Add protease and phosphatase inhibitors see recipe to a final concentration of 1 x Prepare fresh just before use and cool to 4 C Upon addition of orthovanadate the buffer will assume a yellow color Current Protocols in Cell Biology BN lysis buffer stock solution without detergent and inhibitors 2 x 40 ml 1 M bis Tris stock solution pH adjusted to 7 0 with HCl 20 mM final 500 ml 1 M 6 aminohexanoic acid Sigma Aldr
132. blem Cause Solution Protein bands are absent or too faint Background is clear Amount of protein s below Check protein concentration in original detection limit sample Insufficient image development Re stain the gel extending the image development step step 13 until background begins to get too dark Formaldehyde is too old white Use formaldehyde from a fresh polymers in concentrated solution concentrated solution Silver nitrate solution is not fresh Prepare fresh silver nitrate solution just before use Discard the solution if it becomes cloudy Background is too dark Gel has been overdeveloped Monitor image development step 13 continuously Proceed to the next step as soon as the staining is considered satisfactory Poor quality of reagents Use reagents of the highest purity available Prepare the solutions using HPLC grade water Band images and background Concentration of sodium Increase sodium thiosulfate concentration color develop too fast thiosulfate Na2S203 in developer in both solutions e g 2 to 3 fold Do and thiosulfate solutions is not not reuse the solutions optimal Metallic silver is deposited on Powder or dirt deposited on the Handle the gels with clean forceps or the gel surface surface during gel handling gloves that have been rinsed with water Metallic silver present in the silver Protect the solution from light Discard nitrate solution the solution if it gets cloudly 6 6 11 Cu
133. body incubation buffers may help If reprobing is desired blots can be air dried and stored at 4 C for 3 months after chemilu minescence detection After drying store in a sealed freezer bag until use Repeated probing will lead to a gradual loss of signal and in creased background However this will depend in part on the properties of the sample If the primary procedure is problematic due to loss of sensitivity or an increase in the back ground then two possible alternative proce dures for stripping membranes are recom mended The first uses 2 mercaptoethanol and SDS Kaufmann et al 1987 Tesfaigzi et al 1994 Briefly the membranes are incubated in 2 SDS 100 mM Tris Cl pH 7 4 100 mM 2 mercaptoethanol for 30 min at 70 C effec tively removing primary and secondary anti bodies As with the primary procedure recom mended above the repeated probing should be done with caution due to the potential loss of detection signal and 5 nonfat dry milk is required as a blocking agent The milk blocking agent facilitates antibody removal from the blot Kaufmann et al 1987 The second uses guanidine HCl For nylon and PVDF mem branes do not use with nitrocellulose incu bate the immunoblot in 7 M guanidine HCl for Electrophoresis and Immunoblotting 6 2 17 Immunoblotting and Immunodetection 6 2 18 10 min at room temperature The short wash time is critical as guanidine HCl is a very strong denaturan
134. buffer before loading your sample A proper acrylamide concentration for the BN gel and the subsequent second dimension SDS gel should be selected to optimize res olution within the desired molecular weight range Use 4 to 7 BN gels for protein com plexes of more than 1000 kDa 4 to 10 for 500 kDa 4 to 12 for 250 kDa and 4 to 18 for proteins smaller than 100 kDa The first critical step in BN PAGE is the preparation of the sample since potassium and divalent cations that interact with Coomassie blue have to be removed They are substituted by 6 aminohexanoic acid in order to maintain a certain ionic strength of the solution which in creases the solubility of many proteins There fore the lysate has to be dialysed against BN dialysis buffer One frequent problem is that Coomassie blue and Coomassie blue bound proteins pre cipitate in the gel wells and subsequently the sample does not enter the gel In this case either reduce the amount of sample or im prove dialysis of the sample Use a larger dial ysis reservoir volume remove all air bubbles at the dialysis membrane or prolong dialysis time Depending on the detergent the dye front might contain peaks of detergent that prevent proteins from entering below these areas This is pronounced when using polyoxyethylene detergents such as Brij 96 and Triton X 100 If your multiprotein complex of interest runs at a higher position than these precipitates then this mig
135. buffer in the upper chamber decreases a leak has occurred 22 After the bromphenol blue tracking dye has reached the bottom of the separating gel disconnect the power supply Refer to Safety Considerations under Electricity and Electrophoresis Disassemble the gel 23 Discard electrophoresis buffer and remove the upper buffer chamber with the attached gel sandwich 24 Orient the gel so that the order of the sample wells is known remove the sandwich from the upper buffer chamber and lay the sandwich on a sheet of absorbent paper or paper towels 25 Carefully slide one of the spacers halfway from the edge of the sandwich along its entire length Use the exposed spacer as a lever to pry open the glass plate exposing the gel 26 Carefully remove the gel from the lower plate Cut a small triangle off one corner of the gel so the lane orientation is not lost during staining and drying Proceed with protein detection Gradient gels are most easily picked up without tearing from the high concentration end of the gel using gloved fingers Single concentration gels lt 10 can be picked up and placed in fixative but are more easily removed if first immersed in fixative while left on the plate allowing the gel to float off The gel can be stained with Coomassie blue or silver UNIT 6 6 or proteins can be electroeluted electroblotted onto a polyvinylidene difluoride PVDF membrane for sub sequent staining or sequence analysis or tr
136. cations are discussed in the Commentary In the following protocols the volumes of solutions are indicated in gel volumes When staining slab mini gels 10 gel vol correspond to 50 to 80 ml STAINING PROTEIN GELS WITH COOMASSIE BLUE Coomassie brilliant blue R 250 a triphenylmethane anionic dye binds avidly to almost all proteins in either native or denatured states and is widely used for detection of proteins in polyacrylamide gels In this protocol proteins separated by either nondenaturing or SDS PAGE one or two dimensional are fixed and stained by soaking the gel in a Coomassie blue staining solution the unbound dye is subsequently removed by washing with destaining solution to yield blue protein bands on a clear background Materials Polyacrylamide gel containing protein s of interest See UNIT 6 Coomassie blue staining solution see recipe Destaining solution 30 v v methanol 10 v v acetic acid in distilled water store up to 4 months at room temperature Storage solution 7 v v acetic acid 5 v v methanol in distilled water store up to 4 months at room temperature Plastic container with lid pipet tip containers are appropriate for staining mini gels Platform shaker optional CAUTION Glacial acetic acid and methanol are volatile and toxic The destaining and storage solutions should be prepared in a chemical fume hood and gloves should be worn throughout the staining procedure Contribu
137. cessary 6 use of a stacking gel is not necessary 7 total protein load capacity is relatively large 8 extrusion and handling are relatively simple 9 extrusion and handling are very difficult Minigel systems provide rapid separations with moderate resolution Microgels Phastgels are precast gels that are slightly smaller than most minigels 4Use of second dimension gels thicker than 1 5 mm is generally not recommended owing to difficulty with either efficient staining or efficient electroblotting Full size gels provide resolution satisfactory for most applications JGiant gels provide very good resolution Specialized equipment is required such as Investigator 2D ESA Iso Dalt Hoeffer Pharmacia or homemade giant size gel systems ing capacity and hence produce stable pH gra dients In theory any desired pH gradient could be produced by blending ampholytes with ap propriate pK values In practice it is relatively easy to produce pH gradients from pH 3 5 or 4 0 to pH 8 0 but stable soluble gradients outside this range are usually not technically feasible Within these pH limits some manipulation of the gradi ent shape and pH range is possible by blending different amounts of specific pH range ampho lytes For example 0 50 ml of pH 5 7 ampholytes plus 0 25 ml of pH 4 8 ampholytes can be used instead of 0 75 ml of pH 4 8 ampholytes alone to increase the separation distance of proteins in the pH 5 0 to 7 0
138. cially above pH 7 De Spite potential side reactions urea is the most useful IEF additive for maintaining solubility of proteins near their isoelectric points Two dimensional PAGE has become a valu able preparative tool for protein isolation in addi tion to its historical role as an analytical method The sensitivity of many protein analysis methods has improved to the point where one or several spots from two dimensional gels are sufficient for protein identification using mass spectrometry methods Commercially available equipment for run ning two dimensional gels can be divided into four groups based on size microgels Amersham Pharmacia Biotech Phast system minigels e g Bio Rad or Amersham Pharmacia Biotech standard or full sized gels e g Bio Rad or Amer sham Pharmacia Biotech and large or giant gels ESA Investigator 2D gel system or Amer sham Pharmacia Biotech Iso Dalt gel system In general the larger the gel the better the final resolution but as gel size increases so do costs difficulty of gel handling and time require ments Standard size gels provide adequate resolu tion for most applications and are relatively easy to handle A 3 mm soluble ampholyte tube IEF gel or a 0 5 mm x 3 mm x 18 cm Immobiline gel has a total protein capacity of 500 ug for com plex protein mixtures such as whole cell extracts The maximum capacity for any single protein spot is 0 5 to 5 ug depending on the solubilit
139. cies Two types of nylon membrane are used for western transfer neutral e g Pall Biodyne A and positively charged e g Pall Biodyne Current Protocols in Cell Biology B Although the positively charged mem branes have very good protein binding charac teristics they tend to give a higher background These membranes remain positively charged from pH 3 to pH 10 Neutral nylon membranes are also charged having a mix of amino and carboxyl groups that give an isoelectric point of 6 5 Because of their high binding capacity positively charged membranes are popular for protein applications using luminescence Nylon membranes require more stringent blocking steps Here 10 nonfat dry milk in TBS is recommended for chromogenic devel opment During luminescence development however background is a more significant problem Compared to dry milk purified casein has minimal endogenous alkaline phosphatase activity AP activity leads to high background and is therefore recommended as a blocking agent for nitrocellulose PVDF and nylon membranes Positively charged nylon requires much more stringent blocking with 6 w v casein and 1 v v polyvinylpyrrolidone PVP Because nonfat dry milk and casein may contain biotin that will interfere with avidin biotin reactions subsequent steps are done without protein blocking agents when using these systems If background is a problem highly purified casein 0 2 to 6 added to the anti
140. clinical diagnostics of human disor ders Schagger 1995 Schagger et al 1996 In principle BN PAGE can be used to iden tify protein complexes in a given biologi cal sample or to further characterize known protein complexes or protein protein inter actions For the second application it is recommended to first test a series of dif ferent nonionic detergents for their effects on the extractability and stability of the protein protein interaction of interest by co immunoprecipitation UNIT 7 2 followed by SDS PAGE UNIT 6 1 Only a successful co purification indicates that the complex of in terest is stable and abundant enough to be de tected by BN PAGE In BN PAGE the dye Coomassie blue which binds nonspecifically to proteins and is itself negatively charged is used Therefore the electrophoretic mobility of a multiprotein complex is determined by the negative charge of the bound Coomassie blue dye and the size and shape of the complex Coomassie blue does not act as a detergent and preserves the structure of protein complexes In contrast to other native gel electrophoresis systems pro tein complexes are separated independently of isoelectric point and therefore the size of a complex can be estimated In addition the binding of Coomassie blue to proteins reduces their tendency to aggregate during the stacking step of the electrophoresis Following the first dimension BN PAGE a number of subsequent biochemical tec
141. commercially available high molecular weight markers can be used e g from Invitrogen or GE Healthcare Current Protocols in Cell Biology 15 16 17 18 Carefully overlay the samples in each well with the BN cathode buffer Fill the upper inner chamber with BN cathode buffer containing 0 02 w v Coomassie blue as described in Reagents and Solutions and the lower outer chamber with BN anode buffer Connect electrodes If a small gel has been prepared e g using BioRad Protean II or IID run at 100 V if a large gel has been prepared e g using BioRad Protean II xi run at 150 V Continue electrophoresis at the appropriate abovementioned voltage until the sample has entered the separating gel At that point increase voltage to 180 V for small gel or 400 V for large gel Coomassie blue comes into contact with the samples inside the wells during the electro phoresis Remove the BN cathode buffer especially from within the wells after two thirds of the gel run Fill the upper inner chamber with BN cathode buffer low Coomassie blue that contains only 0 002 w v Coomassie blue Continue electrophoresis at 180 V for small gel or 400 V for large gel This procedure ensures that the individual lanes can be identified after the gel is run and can be omitted if a second dimension gel is not to be applied see e g Alternate Protocols 1 and2 or if precipitated material is visible between the stacking and separating gel
142. complexes by two dimensional native electrophoresis Anal Biochem 217 220 230 Schagger H Bentlage H Ruitenbeek W Pfeiffer K Rotter S Rother C Bottcher Purkl A and Lodemann E 1996 Elec trophoretic separation of multiprotein com plexes from blood platelets and cell lines Tech nique for the analysis of diseases with defects in oxidative phosphorylation Electrophoresis 17 709 714 Swamy M Minguet S Siegers G M Alarcon B and Schamel W W 2007 A native antibody based mobility shift technique NAMOS assay to determine the stoichiometry of multiprotein complexes J Immunol Methods 324 74 83 Key References Camacho Carvajal et al 2004 See above Describes the separation of cellular lysates by BN PAGE Schagger and von Jagow 1991 See above Describes for first time BN PAGE and second dimension BN SDS PAGE using solubilized mito chondria Swamy et al 2007 See above Details the NAMOS assay with an extensive discus sion of anticipated results Electrophoresis and Immunoblotting al 6 10 21 Supplement 38
143. con tinuous gel systems can be found in Hames 1990 and Schagger 1994 Critical Parameters The success of a gel separation under non denaturing conditions depends on many fac tors and two of the most important are protein solubility and isoelectric point The protein must be soluble at the pH and the ionic strength of the gel and it must be charged at that pH in order to move into the gel If the protein expe riences a pH below its isoelectric point then it will have a net positive charge and will move to the negative electrode Note that this is the reverse of typical SDS PAGE If the protein experiences a pH above its isoelectric point it will have a net negative charge and will migrate to the positive electrode Solubility is a complex issue Membrane as sociated and other hydrophobic proteins are difficult to separate by nondenaturing electro phoresis Schagger 1994 Nonionic deter gents at concentrations up to 1 and solubiliz ing reagents such as urea 4 to 8 M can be used but these reagents especially urea are likely to alter the protein s conformation and most likely the isoelectric point by exposing previously hidden charged groups If detergent or urea must be included for solubilization the mini mum required to solubilize the protein should be used Schagger 1994 lists several nonionic detergents suitable for solubilization Among the more popular are octylglucoside and CHAPS In general detergents
144. coprotein Blot Stain Kit Molecular Probes containing 50x Pro Q Emerald 300 reagent concentrate in DMF Pro Q Emerald 300 dilution buffer Periodic acid oxidizing solution see recipe Current Protocols in Cell Biology ALTERNATE PROTOCOL Electrophoresis and Immunoblotting 6 8 5 Supplement 16 Fluorescence Detection of Glycoproteins in Gels and on Electroblots 6 8 6 Supplement 16 CandyCane glycoprotein molecular weight standards see recipe sufficient volume for 20 gel lanes SYPRO Ruby protein blot stain Methanol spectroscopy grade optional Glacial acetic acid optional 95 C heat block Polystyrene staining dishes e g weighing boat for minigels or larger containers for larger gels Orbital shaker UV epi illuminator Photographic camera or CCD camera and appropriate filters Additional reagents and equipment for SDS polyacrylamide gel electrophoresis UNIT 6 1 and electroblotting UNIT 6 2 Run gel 1 Prepare the protein samples of interest e g crude protein isolates cell lysates serum partially purified plasma membranes for SDS polyacrylamide gel electrophoresis Typically the protein sample is diluted to 10 to100 ug ml with 2x sample buffer heated for 4 to 5 min at 95 C and then 5 to 10 ul of diluted sample is applied per gel lane for x 10 cm gels Larger gels require proportionally more material For convenience CandyCane glycoprotein molecular weight standards may a
145. crylamide solution see Table 6 1 1 4x phosphate gel buffer see Reagents and Solutions and H O If desired degas under vacuum 5 min to speed polymerization Add 10 ammonium persulfate and TEMED Swirl gently to mix Use immediately The recipes produce 40 ml gel solution which is adequate for one gel of dimensions 1 5 mm x 14 cm x 16 cm or two gels of dimensions 0 75 mm x 14 cm x 16 cm Pall reagents and solutions used in the protocol must be prepared with Milli Q purified water or equivalent Units of numbers in table body are milliliters The desired percentage of acrylamide in the gel solution depends on the molecular size of the protein being separated dMust be freshly made Added just before polymerization the separation conditions i e if gel pH lt protein pl the protein will have a net positive charge if gel pH gt protein pl the protein will be negatively charged 3 Pour gel to 2 cm from the top of the gel mold and insert the comb Avoid trapping air bubbles under the comb teeth Air bubbles will cause small semicircular depressions in the well and lead to distortions in the protein banding pattern 4 Allow gel solution to polymerize 1 to 2 hr Polymerization is indicated by a sharp optical discontinuity around the wells Prepare samples and load the wells 5 Solubilize the protein sample to be analyzed using 5 w v sucrose in water or dilute 1 to 5 mM gel buffer if possible Also pr
146. d Immunoblotting 6 8 11 Supplement 16 Fluorescence Detection of Glycoproteins in Gels and on Electroblots 6 8 12 Supplement 16 teins be electroblotted to membranes first and many glycoproteins transfer relatively poorly In addition detection of glycoproteins after electroblotting is very time consuming com pared with direct detection in gels A recently developed approach to the detection of glyco proteins relies upon the utilization of a new fluorescent hydrazide Pro Q Emerald 300 dye that is affixed to glycoproteins using a standard PAS conjugation mechanism Steinberg et al 2001 The glycols present in glycoproteins are initially oxidized to aldehydes using periodic acid The dye then reacts with the aldehydes on the glycoproteins to generate the fluorescent conjugate A reduction step with sodium metabisulfite or sodium borohydride is not re quired to stabilize the resulting conjugate Critical Parameters All stock solutions should be prepared using deionized water dH2O having a resistance of at least 18 MQ All stock solutions may be stored for up to 6 months at room temperature except when specifically indicated Dilution of the DDAO phosphate stock solution or the Pro Q Emerald 300 dye solution should be performed immediately prior to their use in the staining protocols Both reagents are unstable when stored at room temperature as aqueous solutions The staining methods outlined in this unit
147. d separation but give lower resolution Several alternate protocols are provided for specific applications The first two alternate protocols cover electrophoresis of peptides and small proteins separations that require modification of standard buffers either a Tris tricine buffer system see Alternate Pro tocol 1 or a modified Tris buffer in the absence of urea see Alternate Protocol 2 Continuous SDS PAGE is a simplified method in which the same buffer is used for both gel and electrode solutions and the stacking gel is omitted see Alternate Proto col 3 Other protocols cover the preparation and electrophoresis of various types of gels Current Protocols in Cell Biology 6 1 1 6 1 38 December 2007 Published online December 2007 in Wiley Interscience www interscience wiley com DOI 10 1002 047 1 143030 cb0601s37 Copyright 2007 John Wiley amp Sons Inc UNIT 6 1 Electrophoresis and Immunoblotting eS 6 1 1 Supplement 37 One Dimensional SDS PAGE 6 1 2 Supplement 37 ultrathin gels see Alternate Protocol 4 multiple single concentration gels see Support Protocol 1 gradient gels see Alternate Protocol 5 multiple gradient gels see Support Protocol 2 and multiple gradient minigels see Support Protocol 3 Proteins separated on gels can be subsequently analyzed by immunoblotting UNIT 6 2 autoradiography or phosphor imaging UNIT 6 3 or staining with protein dyes UNIT 6 6 Protein size is de
148. d time consuming since it requires copying re typing or photographic reproduction Copies of digital data can be generated more easily and at reduced costs Manipulation of information is also easier when it is in a digital format While the cut and paste analogy comes from physical documen tation it takes on a new perspective when ap plied digitally Electrophoresis images can be resized cropped and inserted into reports Data can be passed to spreadsheets and statistical packages for analysis and later insertion into notebooks and reports These reports can be passed out via the Internet to colleagues throughout the world A single individual can do all this in a few hours Digital analysis also provides an easier method for handling the data when comparing large numbers of results or large numbers of separate experiments Research that requires comparing the banding patterns on 1000 gels containing 50 lanes each can be an undertaking of heroic proportions if the analysis is per formed manually Database software can dra matically speed the analysis and handle the more mundane tasks leaving the researcher free to interpret the data Accuracy The human eye is an extremely versatile measuring instrument It can handle light inten sities covering a range of nearly nine orders of magnitude and is sensitive to a fairly wide spectrum of light Russ 1995 Yet the eye cannot accurately and reproducibly quantitate density and p
149. d SYPRO Ruby protein gel stain with respect to protein detection in two dimensional gels and identification by peptide mass profiling Electrophoresis 21 3673 3683 Describes optimized methods for protein identifica tion by matrix assisted laser desorption time of flight mass spectrometry after staining gels with SYPRO Ruby dye Current Protocols in Cell Biology Internet Resources http www cbs dtu dk databases OGLYCBASE O GLYCBASE a database of 198 glycoprotein en tries with experimentally verified O glycosylation site information http www glycosuite com GlycoSuite a relational database that curates infor mation from the scientific literature on glycoprotein derived glycan structures their biological sources the references in which the glycan was described and the methods used to determine the glycan struc ture http www expasy ch tools glycomod GlycoMod a software tool designed to find all possible compositions of a glycan structure from its experimentally determined mass http www probes com Molecular Probes commercial Web site containing information about fluorescence detection technolo gies including glycoprotein total protein lipopolysaccharides and nucleic acids Contributed by Wayne F Patton Molecular Probes Inc Eugene Oregon Electrophoresis and Immunoblotting 6 8 15 Supplement 16 Digital Electrophoresis Analysis Gel electrophoresis has become a ubiqui tous me
150. d films containing one emulsion layer e g Kodak BioMax MR are optimized for direct exposure techniques with medium energy radioisotopes e g 4C 35S and P but not H The majority of the B particles emitted by these isotopes cannot pass through the polyester support of double coated films and therefore the emulsion layer on the other side of the film is useless Even though direct exposure with single coated films gives better clarity for medium energy isotopes single coated films often require longer exposure times Fluorography therefore is often used to enhance sensitiv ity The blue light sensitive double coated X Omat AR film is generally used for fluorography with 2 5 diphenyloxazole PPO which emits at 388 nm sodium salicylate which emits at 420 nm and commercial fluorographic solutions and sprays e g from Amersham and DuPont NEN which emit light in the blue end of the spectrum Materials Fixed and dried gel see Support Protocol 1 or filter e g from immunoblotting UNIT 6 2 Developer Kodak developer and replenisher prepared according to the manufacturer s instructions 18 to 20 C Fixer Kodak fixer and replenisher prepared according to the manufacturer s instructions 18 to 20 C Metal film cassette or paper film cassette with particle board supports and metal binder clips Plastic wrap e g Saran Wrap X ray film Trays to hold film processing solutions Clips for hanging film 1 In
151. d to a membrane followed by immunoblotting to visualize the proteins of interest Alternate Protocols 1 and 2 PREPARATION OF CELL LYSATES FOR BLUE NATIVE GEL ELECTROPHORESIS Blue Native BN gel electrophoresis is suitable for the separation of pure proteins as well as complex protein mixtures such as subcellular fractions or cell lysates Sample preparation is one of the critical steps in performing high quality BN gels First the proteins have to be present in soluble and native form Thus the choice of detergent is important and one should start BN experiments by testing several nonionic detergents Current Protocols in Cell Biology SUPPORT PROTOCOL Electrophoresis and Immunoblotting al 6 10 5 Supplement 38 Two Dimensional Blue Native Polyacrylamide Gel Electrophoresis 6 10 6 Supplement 38 for the protein protein complex of interest see Commentary The detergent should be effective enough to extract the proteins from cellular membranes but at the same time mild enough to keep multiprotein complexes intact The most commonly used detergents are given in Reagents and Solutions The proteins must be eluted in native form if the purification procedure includes binding to a matrix as e g in immuno or affinity purifications often referred to as immunoprecipitation see UNIT 7 2 Second the sample has to be prepared without potassium or divalent cations since Coomassie blue and Coomassie blue bound prote
152. d visualized with 4 chloro 1 naphthol 4CN Note how background improves with 66 dilution 43 i be is 18 for murine ascites fluid containing monoclonal antibodies Ten to one hundred fold higher dilutions can be used with alkaline phosphatase or luminescence based detection sys tems Both primary and secondary antibody solutions can be used at least twice but long term storage i e gt 2 days at 4 C is not recommended 3 Open bag and pour out blocking buffer Replace with diluted primary antibody and incubate 30 min to hr at room temperature with constant agitation Usually 5 ml diluted primary antibody solution is sufficient for two to three membranes 14 x 14 cm size Incubation time may vary depending on conjugate used When using plastic trays the primary and secondary antibody solution volume should be increased to 25 to 50 ml For membrane strips incubation trays with individual slots are recommended Typically 0 5 to 1 ml solution slot is needed 4 Remove membrane from plastic bag with gloved hand Place in plastic box and wash four times by agitating with 200 ml TTBS nitrocellulose or PVDF or TBS nylon 10 to 15 min each time 5 Dilute secondary antibody HRPO or AP anti Ig conjugate in blocking buffer Commercially available enzyme conjugated secondary antibody is usually diluted 1 200 to 1 2000 prior to use Harlow and Lane 1988 6 Place membrane in new heat sealable plast
153. ddition some proteins simply do not bind well to a particular matrix By using several membrane sheets in place of one the protein can be detected as it passes through each consecutive sheet This will give an indication of how efficiently the membrane binds to a particular protein 14 Proceed with immunoprobing and visual detection of proteins see Basic Protocols 2 and 3 and Alternate Protocols 3 and 4 ALTERNATE PROTEIN BLOTTING WITH SEMIDRY SYSTEMS Beek ees Even and efficient transfer of most proteins is also possible with semidry blotting a convenient alternative to tank transfer systems Instead of being placed vertically into a tank filled with transfer buffer the gel is held horizontally between buffer saturated blotting paper that is in contact with the electrodes Fig 6 2 2 greatly reducing the amount of buffer required The electrodes are close together giving high field strengths and rapid transfer with a standard electrophoresis power supply Prolonged transfers gt 1 hr are not recommended tank blotting see Basic Protocol 1 should be used for proteins that require long blotting times for efficient transfer Additional Materials also see Basic Protocol 1 Six sheets of Whatman 3MM filter paper or equivalent cut to size of gel and saturated with transfer buffer Semidry transfer unit Amersham Pharmacia Biotech Bio Rad or Sartorius 1 Prepare samples and separate proteins using small or standard sized one or two
154. delines imply the majority of pro teins have isoelectric points between pH 4 0 and 8 0 There are however many exceptions A protein with a highly acidic isoelectric point e g pepsin with a pI of 2 2 will remain negatively charged at a pH down to its pl Although a full range of pH options are given extremes of pH lt 4 0 and gt 9 0 should be avoided if possible to minimize denaturation or inactivation By picking an appropriate elec trophoresis pH it is possible to ensure that the protein of interest will be either positively or negatively charged so that it can be selectively run into the gel excluding a large proportion of contaminants that have the opposite or no charge Furthermore the pH conditions deter mine the mobility and can be adjusted to ensure a difference in mobility between the protein of interest and contaminants Continuous gel systems see Basic Protocol offer the most flexibility in terms of separation design The pH can be tailored so that a given protein has a net positive neutral or negative charge Depending on the polarity of the gel the protein can then be excluded from or elec trophoresed into the gel Discontinuous gels have a fixed pH and gel polarity For the non denaturing Laemmli gel presented in the Alter nate Protocol the proteins of interest should have an isoelectric point of 7 0 in order to be negatively charged so that they move into the gel Other more basic and more acidic dis
155. diago nal because internal disulfides can produce a more compact molecular shape causing faster migra tion in the first dimension Investigator 2D gel system The Investigator 2D gel system was intro duced in 1990 by Millipore as the first commer cial large or giant format two dimensional gel system designed for analytical purposes al though analogous homemade units had been re ported earlier Garrels 1979 Young et al 1983 This product line including precast first and second dimension gels can now be purchased from ESA The Investigator 2D gel system uses larger gel sizes than the gels described in this unit as well as a number of novel approaches and reagents designed to enhance resolution and gel to gel reproducibility The major features of this system include the following an increased length of the first dimension gel 20 cm an increased length and width of the second dimension gel 20 x22 cm a thread reinforcement of the isoelectric focusing gel temperature control during electro phoresis of the second dimension gel and use of a special high tensile strength acrylamide to fa Current Protocols in Cell Biology cilitate handling of the large thin second di mension gel Narrow gel tubes 1 2 mm for the first dimension are standard but 3 mm inner diameter tubes are available to accommodate larger protein loads The 3 mm IEF gels do not have the thread reinforcement One modifica tion related
156. disulfide bond within the linker region This unit also includes support protocols describing pI standards and pH profile meas urements see Support Protocol 1 electrophoresis of immobilized pH gradient gels see Support Protocol 2 casting Immobiline gels see Support Protocol 3 preparation of tissue culture cells and solid tissues for isoelectric focusing see Support Protocols 4 and 5 preparation of molecular weight standards for two dimensional gels see Support Protocol 6 and two dimensional protein databases see Support Protocol 7 Contributed by Sandra Harper Jacek Mozdzanowski and David Speicher Current Protocols in Cell Biology 1999 6 4 1 6 4 36 Copyright 1999 by John Wiley amp Sons Inc UNIT 6 4 Electrophoresis and Immunoblotting 6 4 1 Supplement 4 BASIC PROTOCOL 1 Two Dimensional Gel Electrophoresis 6 4 2 Supplement 4 NOTE High purity water e g Milli Q water or equivalent is essential for all solutions For cautions relating to electricity and electrophoresis see Safety Considerations in the introduction to UNIT 6 1 HIGH RESOLUTION EQUILIBRIUM ISOELECTRIC FOCUSING IN TUBE GELS This protocol describes the preparation of broad range first dimension gels using soluble ampholytes that resolve proteins with pI values between approximately 4 0 and 8 0 and is based on the original procedure described by O Farrell 1975 The procedure presented here refers specificall
157. ds are likely to be 20 to 100 ug for silver staining and 200 to 1000 ug for Coomassie blue staining The salt concentration in samples should be kept lt 50 mM and if the sample contains SDS the final SDS concentration should be lt 0 25 17 Place the lid on the Multiphor II unit and connect the leads to a power supply Focus the gels with constant voltage for 2 to 3 hr at 500 V followed by 12 to 16 hr at 3500 V for a total of 40 to 60 k Vhr Refer to the user manual for exact recommended voltage conditions for each type of Immobiline DryStrip The optimal number of Vhr will depend upon the pH range of the Immobiline Strip used the type of sample and the sample load and volume therefore the optimal Vhr should be empirically determined for different applications 18 When isoelectric focusing is complete disconnect the power supply and remove the cover from the Multiphor IJ unit Remove the electrodes electrode strips and sample cup bar from the tray If gels are to be run in the second dimension immediately after isofocusing steps 1 to 3 of Basic Protocol 4 should be completed prior to terminating isofocusing 19 Using forceps remove the DryStrips from the tray If the gels are to be run in the second dimension immediately place in a petri dish with the support film along the wall of the dish and proceed directly to equilibration of the gel see Basic Protocol 4 step 4 Alternatively gels may be stored sealed in a plastic bag a
158. e 10x Triton X 100 Prepare a 10 w v solution of Triton X 100 in water Store up to 1 year possibly longer at room temperature 10x NP 40 Prepare a 10 w v solution of Nonidet P 40 NP 40 in water Store up to 1 year possibly longer at room temperature 10x dodecylmaltoside Prepare a 10 w v solution of dodecylmaltoside Sigma Aldrich in water Store up to 1 year possibly longer at 4 C Electrophoresis Any nonionic detergent that has been proven to be useful for the cells and proteins and of interest can be used as well Immunoblotting 6 10 15 Current Protocols in Cell Biology Supplement 38 Two Dimensional Blue Native Polyacrylamide Gel Electrophoresis 6 10 16 Supplement 38 High percentage BN separating gel solution 5 ml 3x BN gel buffer see recipe 5 63 ml acrylamide bisacrylamide mix see recipe 4 38 ml 70 v v glycerol 42 ul 10 w v aqueous ammonium persulfate add immediately before pouring gel 4 2 ul TEMED add immediately before pouring gel This will result in a 15 solution Adjust volumes and acrylamide bisacrylamide concen tration as necessary Useful concentrations range from 6 to 18 Low percentage BN separating gel solution 5 ml 3x BN gel buffer see recipe 1 5 ml acrylamide bisacrylamide mix see recipe 8 5 ml H2O 54 ul 10 w v aqueous ammonium persulfate add immediately before pouring gel 5 4 ul TEMED add immediately before pouring gel This will resul
159. e at least 10 ml dialysis buffer per 100 ul sample Fix the tube upside down inside the beaker and remove air bubbles from the hole beneath the cap Make sure that the dialysis membrane is not damaged Dialyze lysate 18 19 Switch on the magnetic stirrer and dialyze for 6 hr or overnight in a cold room Make sure that stirring is not creating air bubbles at the dialysis membrane Collect the dialysed cell lysate in a new chilled microcentrifuge tube and analyze by BN PAGE Basic Protocol 1 If the sample will be subjected to two dimensional analysis reserve one third of the sample to serve as a control in the second dimension analysis Freezing and thawing of cell lysates might lead to the aggregation of proteins Therefore the cell lysate should be separated immediately by BN PAGE For some proteins freezing might be possible and has to be determined empirically Current Protocols in Cell Biology Electrophoresis and Immunoblotting ee 6 10 7 Supplement 38 BASIC PROTOCOL 2 Two Dimensional Blue Native Polyacrylamide Gel Electrophoresis 6 10 8 Supplement 38 SECOND DIMENSION DENATURING ELECTROPHORESIS A denaturing second dimension gel is suitable to identify and characterize multiprotein complexes It is recommended to use vertical denaturing SDS discontinuous gel elec trophoresis Laemlli method of SDS PAGE described in detail in UNIT 6 1 Basic Protocol 1 These gels can be linear or gr
160. e gels A systematic analysis Electrophoresis 6 427 448 Rabilloud T Vuillard L Gilly C and Lawrence J J 1994 Silver staining of proteins in polyacrylamide gels A general overview Cell Mol Biol 40 57 75 Righetti P G and Drysdale J W 1974 Isoelectric focusing in gels J Chromatogr 98 271 321 Rosenfeld J Capdevielle J Guillemot J C and Ferrara P 1992 In gel digestion of proteins for internal sequence analysis after 1 dimensional or 2 dimensional gel electrophoresis Anal Bio chem 203 173 179 Schagger H and von Jagow G 1987 Tricine so dium dodecyl sulfate polyacrylamide gel elec trophoresis for the separation of proteins in the range from 1 to 100 kDa Anal Biochem 166 368 379 Steinberg T H Jones L J Haugland R P and Singer V L 1996 SYPRO Orange and SYPRO Red protein gel stains One step fluorescent staining of denaturing gels for detection of nanogram levels of protein Anal Biochem 239 223 237 Switzer R C Merril C R and Shifrin S 1979 A highly sensitive silver stain for detecting protein and peptides in polyacrylamide gels Anal Bio chem 98 231 237 Wallace A and Saluz H P 1992 Ultramicrodetec tion of proteins in polyacrylamide gels Anal Biochem 203 27 34 Wilm M Shevchenko A Houthaeve T Breit S Schweigerer L Fotsis T and Mann M 1996 Femtomole sequencing of proteins from polyacrylamide gels by nano electrospray mass s
161. e temperature of the gel to 0 to 4 C for a few hours faster and more sensitive methods in volve the formation of insoluble complexes with heavy metal salts such as copper Garfin 1990 or zinc Fernandez Patron et al 1995a A protocol for rapid reversible staining with zinc is described in Basic Protocol 4 The pro tocol uses a kit that is commercially available at a reasonable price If cost is a major issue however the reader is referred to the protocol described by Fernandez Patron et al 1995a Additional staining methods not discussed in this unit include radiolabeling with gt gt S thiourea and silver bromide Wallace and Saluz 1992 combined Coomassie blue zinc staining Fernandez Patron et al 1995b and mixed Evans blue rhodamine B staining Na et al 1994 Critical Parameters Comparison of staining procedures None of the staining methods is optimal for all types of applications The choice of the most appropriate method for a particular experiment will depend on a number of considerations including sensitivity simplicity duration long term documentation quantitation and downstream applications The following guide lines are restricted to the procedures described in this unit namely staining with Coomassie blue Basic Protocol 1 and Alternate Protocol 1 silver Basic Protocol 2 SYPRO Ruby Basic Protocol 3 and zinc Basic Protocol 4 Current Protocols in Cell Biology Sensitivity Figure
162. e Ferguson plots Rodbard and Chrambach 1971 An drews 1986 For example if two components differ in size but have the same charge per unit size e g for a multimeric protein with identi cal subunits curves similar to those illustrated by BSA monomer and dimer Fig 6 5 2A will result Note that when the curve is extrapolated back to 0 T it is evident that the monomer and the dimer have similar free solution mobili ties Furthermore as the acrylamide concentra tion is increased the separation between the two also increases However if two proteins have similar sizes but different amounts of charge the curves will be parallel on the log plot This is illustrated by the carbonic anhy drase isoforms Fig 6 5 2B In this example optimal separation of the isoforms occurs at the lower concentrations of acrylamide as this is a log plot Further applications of nondenaturing elec trophoresis include preparative purification The pH of the gel determines the net charge on the protein Below its isoelectric point pI a protein will have a net positive charge whereas above its pI it will have a net negative charge Electrophoresis and Immunoblotting 6 5 9 Supplement 5 One Dimensional Electrophoresis Using Nondenaturing Conditions 6 5 10 Supplement 5 In general most proteins will be positively charged at pH 2 0 to 4 0 above pH 8 0 most proteins will be negatively charged As these general gui
163. e caster The plug is removed when casting gradient gels see Support Protocol 2 Current Protocols in Cell Biology Table 6 1 9 Recipes for Multiple Single Concentration Polyacrylamide Gels SEPARATING GEL Stock solution Final acrylamide concentration in the separating gel 5 6 7 8 9 10 11 12 13 14 15 30 w v acrylamide 52 62 72 83 93 103 114 124 134 145 155 0 8 w v bisacrylamide 4x Tris Cl SDS 78 78 78 78 78 78 78 78 78 78 78 pH 8 8 H20 181 171 160 150 140 129 119 109 98 88 78 10 w v ammonium 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 persulfate TEMED 0 21 0 21 0 21 0 21 0 21 0 21 0 21 0 21 0 21 0 21 0 21 Preparation of separating gel In a500 ml side arm flask mix 30 acrylamide 0 8 bisacrylamide solution see Table 6 1 1 4x Tris Cl SDS pH 8 8 Table 6 1 1 and H20 Degas under vacuum 5 min Add 10 ammonium persulfate and TEMED Swirl gently to mix Use immediately STACKING GEL In a 250 ml side arm flask mix 13 0 ml 30 acrylamide 0 8 bisacrylamide solution 25 ml 4x Tris Cl SDS pH 6 8 Table 6 1 1 and 61 ml H20 Degas under vacuum 5 min Add 0 25 ml 10 ammonium persulfate and 50 ul TEMED Swirl gently to mix Use immediately The recipes produce about 300 ml of separating gel and 100 ml of stacking gel which are adequate for ten gels of dimensions 1 5 mm x 14 cm x 14 cm with extra solution should there be a leak or spill while casting the gels For thinner sp
164. e centrifuge with adaptor cavities for microcentrifuge tubes in rotor accommodating 50 ml tube Dialysis membranes MWCO 10 to 50 kDa boiled and kept at 4 C in 0 001 M EDTA 1 ml reaction tubes e g microcentrifuge tubes Beaker 100 ml to 1 liter depending on sample size Harvest and wash the cells 1 Place cell culture dish containing cells on ice 2 For adherent cells wash cells three times each time with 2 to 10 ml ice cold 1x PBS Cells grown in suspension are washed three times with ice cold 1x PBS by repeatedly pelleting the cells by centrifugation for 5 min at 350 x g 4 C The volume of PBS used for one washing step should be equal to the volume of medium in which the cells were cultivated 3 Add 2 to 10 ml i e volume in which cells were cultivated of ice cold 1x PBS to the dish remove the cells using a cell scraper and transfer the cell suspension to a 10 to 50 ml centrifuge tube This step is omitted for cells grown in suspension Current Protocols in Cell Biology Collect cells by centrifugation for 5 min at 350 x g 4 C and remove supernatant Resuspend cells in 1 ml cold 1x PBS transfer cell suspension into a small reaction tube e g microcentrifuge tube and pellet cells by centrifugation for 5 min at 350 x g 4 C Remove and discard the supernatant using a pipet Either move on to step 8 or freeze the cell pellet at 20 C Frozen cells can be stored at 20 C for at least
165. e companies e g Bio Rad offer different screens for use with different isotopes They vary principally in the protective coating on the screen which is optimized for low or high energy B particles or yrays No coating is typically used for weak B emitters such as tritium More recently screens have also been developed that measure chemilumines cence Such screens are particularly valuable for use with many nonradioactive labeling protocols The protocol below is for the PhosphorImager system from Molecular Dynamics other phosphor imaging systems are available from Bio Rad Imaging Research and National Diagnostics 105 3 4_ Q 3 10 2 5 O 3 103 2 3 Z S E 102 1 5 5 2 E ro 1 g g 10 1 5 E a 109 4 05 1071 a a L 101 102 103 104 105 106 107 108 Disintegrations mm2 Figure 6 3 2 32P dilution series quantified on Model GS 525 phosphor imager Squares com pared to film circles Image courtesy of Bio Rad Hercules Calif Current Protocols in Cell Biology Materials Gel or filter e g from immunoblotting UNIT 6 2 PhosphorImager system Molecular Dynamics including ImageEraser light box Exposure cassette with phosphor screen Scanning software 1 Erase any latent image on the phosphor screen left by a previous user or caused by background radiation by exposure to visible light The PhosphorImager system comes with an extra bright light box ImageEraser for
166. e dimensional image with spots treated as hills and background as valleys A large number of Gaussian curves are then combined to describe the topology of the im age Many other methods make use of a digi tal imaging technique known as filtering In essence filtering is a way to weight the value of a pixel and its neighbors in order to generate anew value fora pixel By passing a filter across an image pixel by pixel a secondary image is generated Filters can be designed for many tasks including sharpening an image or remov ing high frequency noise Filters can also be generated to help detect spots by making im ages that are first and second derivatives of the original image The derivative images indicate inflection points in the intensity pattern and can be used to detect spot centers and edges In a different method called thresholding filters can be used to detect the edges of objects Instead of looking for inflection points thresh old filters identify intensities above a set level or at ratios between central and edge pixels above a set value Since the edges on two di mensional spots tend to be diffuse sharpening filters are sometimes used prior to the thresh olding filter In some cases multiple techniques are used to detect spots Glasbey and Horgan 1994 Current Protocols in Cell Biology Unlike one dimensional detection detec tion on images of two dimensional experi ments usually requires secondary processing
167. e g Saran Wrap Gel dryer with vacuum pump After electrophoresis remove the gel and the supporting glass plates from the electrophoresis apparatus and place in a glass dish Carefully remove the upper glass plate by gently prying apart the corners with a metal spatula Make a notch in the upper right hand corner of the gel for orientation Since the gel contains radiolabeled proteins be sure to follow the necessary guidelines for handling radioactivity Everything that comes in contact with the gel is potentially radio active For gels with lt 15 acrylamide and lt 1 5 mm thick Place the glass dish in a fume hood and pour enough fixing solution into the dish to cover the gel Place the dish on a rotary shaker and gently rotate until 5 min after all of the blue color from the bromphenol blue in the sample buffer if used has disappeared 30 min total The bromphenol blue typically used in SDS PAGE sample buffer will turn yellow as the acidic fixing solution diffuses into the gel During fixing the gel will typically slide off the lower glass plate which can be removed For gels with 215 acrylamide or gt 1 5 mm thick Fix gel as in step 2a but soak 1 hr in alternative fixing solution The glycerol in the alternative fixing solution should help prevent cracking during drying Pour off the fixing solution and rinse the gel for a few minutes with deionized water CAUTION Remember that solutions that come in contact with
168. e gradient gels requires a peristaltic pump and a multiple gel caster Gel solution is introduced through the bottom of the multiple caster 2 Set up the peristaltic pump Fig 6 1 3 Using a graduated cylinder and water adjust the flow rate so that the volume of the gradient solution plus volume of plug solution is poured in 15 to 18 min 25 ml min 3 Setup the gradient maker Close all valves and place a stir bar in the mixing chamber which is the one with the outlet port Attach one end of a piece of Tygon tubing to the outlet of the gradient maker Run the other end of the tubing through the peristaltic pump and attach it to the red inlet port at the bottom of the caster Choose a gradient maker that holds no more than four times the total volume of the gradient solution to be poured i e a 1000 or 500 ml gradient maker 4 Prepare solutions for the gradient maker Table 6 1 11 Deaeration is not recommended for gradient gels 5 Immediately after adding TEMED to the gel solutions pour the light low concentration solution into the mixing chamber the one with the port Open the mixing valve slightly to allow the tunnel to fill and to avoid air bubbles Close the valve again and pour the heavy high concentration acrylamide solution into the reservoir chamber 6 Start the magnetic stirrer and open the outlet valve then start the pump and open the mixing valve In units for casting multiple gels acrylamide solution
169. e image 6 Immerse the film for 5 min in 18 to 20 C fixer then wash for 15 min in running water 7 Hang the film to dry The orientation of the film with respect to the gel can be determined by the images of the fluorescent markers FIXING AND DRYING GELS FOR AUTORADIOGRAPHY SUPPORT SDS PAGE gels containing radiolabeled proteins should be fixed and dried before PROTEGE exposure to film This will prevent the gel from sticking to the film improve the sharpness of the image and increase sensitivity slightly However if the specific activity of the sample is high or the detection method is sensitive e g where a phosphor imager is used see Alternate Protocol 2 then fixing and drying the gel may not be necessary Gel dryers are available from a number of manufacturers e g Bio Rad most of which use heat and a vacuum to accelerate the drying process Electrophoresis and Immunoblotting 6 3 3 Current Protocols in Cell Biology Detection and Quantitation of Radiolabeled Proteins in Gels and Blots 6 3 4 Materials 2a 2b Gel from SDS PAGE UNIT 6 1 Fixing solution 10 v v glacial acetic acid 20 v v methanol in H O Alternative fixing solution for gels with 215 acrylamide or thicker than 1 5 mm 3 v v glycerol 10 v v glacial acetic acid 20 v v methanol in H O Glass dish Rotary shaker Filter paper Whatman 3MM in sheets at least 1 to 2 cm larger than gel Plastic wrap
170. e proteins in the gel is accomplished with the use of an agarose gel that is modified by the addition of glycidol to yield a glyceryl agarose that con tains aldehyde groups after oxidation by peri odate Proteins are covalently bound in the gel after electrophoresis by reaction of their amino groups with the aldehydes in the presence of the reducing agent sodium cyanoborohydride Further direct probing with antibodies can be carried out without the need for transfer to Current Protocols in Cell Biology another support Shainoff 1993 Composite gels of agarose or glyoxal agarose have also been prepared to provide differing degrees of sieving Peacock and Dingman 1968 Shainoff 1993 Although few proteins are as large as VWF the evaluation of von Willebrand factor mul timers illustrates an important clinical applica tion of the use of agarose as a medium for electrophoresis Assessment for the presence of the largest multimers is physiologically impor tant for the diagnosis of von Willebrand disease and for selection of the most appropriate treat ment Rick 2001 The original procedure for this analysis see Alternate Protocol with mi nor modifications included glycidol in the agarose which served to aid in the fixation of the protein bands while the further washing and antibody identification steps were accom plished Hoyer and Shainoff 1980 It was subsequently found that diffusion of protein bands was not a limiting
171. e solubilized by boiling in the presence of SDS an aliquot of the protein solution is applied to a gel lane and the individual proteins are separated electrophoretically The stacking gel through a combination of low porosity and a lower buffer concentration and pH concentrates proteins into a thin stack before they enter the resolving gel 2 Mercaptoethanol 2 ME or dithiothreitol DTT is added during solubilization to reduce disulfide bonds This protocol is designed for a vertical slab gel with amaximum size of 0 75 mm x 14cm x 14cm For thicker gels or minigels see Basic Protocol 2 and Support Protocol 3 the volumes of stacking and separating gels and the operating current must be adjusted Additional protocols describe the preparation of ultrathin gels see Alternate Protocol 4 and gradient gels see Alternate Protocol 5 as well as the use of gel casters to make multiple gels both single concentration gels see Support Protocol 1 and gradient gels see Support Protocol 2 Materials Separating and stacking gel solutions Table 6 1 1 H2O saturated isobutyl alcohol 1x Tris Cl SDS pH 8 8 dilute 4x Tris Cl SDS pH 8 8 Table 6 1 1 Protein sample on ice 2x and 1x SDS sample buffer see recipe Protein molecular weight standards Tables 6 1 2 and 6 1 3 6x SDS sample buffer see recipe optional 1x SDS electrophoresis buffer see recipe Current Protocols in Cell Biology Electrophoresis apparatus e g Protean II 16 c
172. ecular Biochemi cals Both dyes were excited using the instrument s 300 nm UV B transilluminator and images were captured using the instrument s cooled CCD camera Pro Q Emerald 300 dye signal was collected using the standard 520 nm band pass emission filter Gels were then stained with SYPRO Ruby protein gel stain and SYPRO Ruby dye signal was collected using the 600 nm band pass emission filter provided with the instrument Figure courtesy of Courtenay Hart Molecular Probes Stain the gel for total protein 12 In order to counter stain non glycosylated proteins in the sample pour the SYPRO Ruby protein gel staining solution into a small clean plastic dish For one or two standard size mini gels use 50 ml to 100 ml of staining solution for larger gels use 500 to 750 ml 13 Place the gel into the staining solution and gently agitate e g on an orbital shaker at 50 rpm at room temperature The staining time ranges from 90 min to 3 hr depending upon the thickness and percentage of polyacrylamide in the gel Specific staining can be seen in as little as 30 min However a minimum of 3 hr of staining is required for the maximum sensitivity and linearity For convenience gels may be left in the dye solution overnight or longer without over staining Current Protocols in Cell Biology 14 After staining rinse the gel in water for 30 to 60 min to decrease background fluorescence Alternatively to further decrease background fluor
173. ed in 2 v v glycerol for another 15 to 30 min placed onto Whatman 3 MM filter paper and then dried on a vacuum system FLUORESCENCE DETECTION OF PROTEINS IN GELS In this method proteins separated on polyacrylamide gels either nondenaturing UNIT 6 5 or SDS PAGE one or two dimensional UNIT 6 1 or UNIT 6 4 are incubated with SYPRO Ruby a commercially available fluorescent compound that interacts specifically with proteins Following incubation protein bands can be readily visualized using standard 300 nm transillumination Materials Polyacrylamide gel containing protein s of interest See UNIT 6 1 UNIT 6 4 OF UNIT 6 5 SYPRO Ruby protein gel stain Molecular Probes Distilled water Plastic container with lid pipet tip containers are appropriate for staining mini gels Platform shaker optional 300 nm UV transilluminator Photographic camera or CCD camera optional CAUTION The potential toxicity of the SYPRO Ruby dye which comprises an organic component and ruthenium has not been fully evaluated Gloves should be worn through out the staining procedure For disposal the stain solution should be poured through activated charcoal and the dye adsorbed to activated charcoal destroyed in a chemical incinerator following local environmental regulations NOTE Protect SYPRO Ruby protein gel stain from light If the plastic container with lid used for staining is transparent cover it completely with aluminum foil Cu
174. els are also run at 3000 V but may require a focusing time of 4 to 7 hr 16 After removing gels from the electrophoresis apparatus detect proteins using any conventional staining technique such as Coomassie blue or silver staining 17 Preserve the gels by sealing in a plastic bag or by drying for a permanent record Alternatively electrotransfer the proteins on the gel to a membrane To dry a gel presoak it first in a preservation solution For silver stained gels use a solution of 5 to 10 w v glycerol 30 v v ethanol for Coomassie blue stained gels use a solution of 5 to 10 w v glycerol 16 v v ethanol 8 w v acetic acid After soaking the gel place it on a glass plate gel side up cover with a cellophane sheet soaked in preservation solution and allow to dry at room temperature For electrotransfer UNIT 6 2 use film remover to remove the plastic support film from the gel Electrotransfer of proteins to a polyvinylidene difluoride PVDF membrane using a Multiphor II NovaBlot transfer kit Amersham Pharmacia Biotech is recommended Transferring IPG gels requires special procedures see the transfer kit manual for instruc tions CASTING AN IMMOBILINE GEL An alternative to precast IPG gels is the use of Amersham Pharmacia Biotech Immobilines to cast immobilized pH gradient gels with customized pH gradients and ranges including very narrow pH ranges to improve separation of proteins with small charge differences
175. ent gel and then running it in a vertical SDS sec ond dimension gel Isoelectrofocusing is per formed in a horizontal electrophoresis unit in which multiple gel strips may be run simulta neously Equipment and reagents are also avail able for the user to cast custom Immobiline gels in the laboratory Some of the major advantages of using precast IPG gels are their ease of use and high reproduci bility the time savings realized and the fact that precision narrow range gradients can be used to resolve small charge differences Because the pH gradient is covalently coupled to the polyacry lamide gel matrix the pH gradient remains stable and linear during prolonged electrophoresis thus ensuring reproducibility This is in contrast to conventional IEF gels where gradient drift occurs during prolonged electrophoresis Additionally the precast gels can be rehydrated in water or in solutions with one or more additives such as urea CHAPS or Triton X 100 carrier ampholytes glycerol and reducing agents which may help to increase protein solubility Precast gel strips are available with pH ranges such as pH 3 0 to 10 0 or pH 4 0 to 7 0 as well as narrower ranges as DryPlates In addition a variety of Immobilines permits the user to cast IPG gels with customized pH gradients of any gradient range and shape between pH 3 0 and pH 10 0 Table 6 4 3 lists types of commercially avail able gels and Immobilines With the Immobiline Current
176. ent for SDS polyacrylamide gel electrophoresis UNIT 6 1 electroblotting procedures UNIT 6 2 and SYPRO Ruby protein blot staining see Alternate Protocol Run gel 1 Prepare the protein samples of interest e g crude protein isolates cell lysates serum partially purified plasma membranes for SDS polyacrylamide gel electrophoresis UNIT 6 1 Typically the protein sample is diluted to 10 to 100 ug ml with 2x sample buffer heated for 4 to 5 min at 95 C and 5 to 10 ul of diluted sample is then applied per gel lane for 8x 10 cm gels Larger gels require proportionally more material Current Protocols in Cell Biology 2 For convenience CandyCane glycoprotein molecular weight standards may also be applied to a lane or two Typically 2 ul of this standard is diluted in 6 ul of sample buffer and heated in the same manner as the samples to be characterized These standards contain a mixture of glycosylated and non glycosylated proteins ranging from 14 to 180 kDa in molecular weight The standards serve as molecular weight markers and as alternating bands of positive and negative controls for glycoprotein and total protein detection Each protein is present at 0 5 mg ml Separate proteins by SDS polyacrylamide gel electrophoresis using standard meth ods UNIT 6 1 The procedure is optimized for gels that are 0 5 to 1 mm thick Blot proteins 3 4 5 After electrophoresis transfer the proteins to PVDF membrane using
177. epare native protein standards The concentration of protein will vary depending on the sample complexity and detection method For Coomassie blue staining of highly enriched samples such as the standards use I to 2 mg ml 1 to 2 ug ul For more complex mixtures use 5 to 10 mg ml 5 to 10 ug ul Load 10 to 100 fold less for silver staining In general samples should be loaded in a minimum volume preferably 10 to 20 ul for 0 75 and 1 5 mm thick gels respectively With thin gels this means using a more concentrated protein sample 6 Remove comb carefully and rinse wells with electrophoresis buffer appropriate 4x gel buffer diluted to 1x Rinsing with electrophoresis buffer is needed to remove residual unpolymerized acrylamide monomer which will continue to polymerize after comb removal creating uneven wells that may interfere with sample loading Current Protocols in Cell Biology Electrophoresis and Immunoblotting 6 5 3 Supplement 5 One Dimensional Electrophoresis Using Nondenaturing Conditions 6 5 4 Supplement 5 Table 6 5 3 Recipes for Tris Nondenaturing Polyacrylamide Gels pH Range 7 1 to 8 9 Final acrylamide concentration in gel 5 75 10 12 5 15 17 5 20 Stock solution 30 acrylamide 0 8 6 7 10 13 3 16 8 20 23 32 26 6 bisacrylamide 4x Tris gel buffer 10 10 10 10 10 10 10 H O 23 08 19 78 1648 12 98 9 78 646 3 18 10 w v ammonium 0 2 0 2 0 2 0 2 0 2 0 2 0 2 persu
178. equencing recrystallization is useful Commercially available electrophoresis grade SDS is usually of sufficient purity for most applications continued Current Protocols in Cell Biology Add 100 g SDS to 450 ml ethanol and heat to 55 C While stirring gradually add 50 to 75 ml hot H20 until all SDS dissolves Add 10 g activated charcoal Norit 1 Sigma to solution After 10 min filter solution through Whatman no 5 paper on a Buchner funnel to remove charcoal Chill filtrate 24 hr at 4 C and 24 hr at 20 C Collect crystalline SDS on a coarse frit porosity A sintered glass funnel and wash with 800 ml 20 C ethanol reagent grade Repeat crystallization without adding activated charcoal Dry recrystallized SDS under vacuum overnight at room temperature Store in a desiccator over phosphorous pentoxide P205 in a dark bottle If proteins will be electroeluted or electroblotted for protein sequence analysis it may be desirable to crystallize the SDS twice from ethanol H2O Hunkapiller et al 1983 SDS electrophoresis buffer 5 x 15 1 g Tris base 0 125 M final 72 0 g glycine 0 96 M final 5 0 g SDS 0 5 w v final recrystallization see recipe optional H20 to 1000 ml Dilute to 1x or 2x for working solution as appropriate Do not adjust the pH of the stock solution as the solution is pH 8 3 when diluted Store at 0 to 4 C until use up to 1 month SDS sample buffer 2x for discontinuous systems 25 ml 4x T
179. er Extra glass or acrylic plates or polycarbonate sheets are used to fill any free space in the caster and to ensure that the gel sandwiches are held firmly in place The multiple casters from Hoefer have a notch in the base designed for casting gradient gels A silicone rubber insert fills up this space when casting single concentration gels The Hoefer spacers are T shaped to prevent slipping The flanged edge of the spacer must be positioned against the outside edge of the glass plate Placing a sheet of wax paper between the gel sandwiches will help separate the sandwiches after polymerization 2 Fit the gel sandwiches tightly in the multiple gel caster Use an acrylic plate or polycarbonate separation sheet to eliminate any slack in the chamber Loosely fitting sandwiches in the caster will lead to unevenly cast gels creating distortions during electrophoresis 3 Place the front faceplate on the caster clamp it in place against the silicone gasket and verify alignment of the glass plates and spacers 4 Prepare the separating gel solution as directed in Table 6 1 1 For five 0 75 mm thick gels prepare 30 ml solution i e double the volumes listed To compute the total gel volume needed multiply the area of the gel e g 7 3 x 8 3 cm by the thickness of the gel e g 0 75 mm and then by the number of gels in the caster If needed add 4 to 5 ml of extra gel solution to account for the space around the outside of the gel sand
180. er see recipe Powder free gloves Scotch Brite pads 3M or equivalent sponge Whatman 3MM filter paper or equivalent Transfer membrane 0 45 um nitrocellulose Millipore or Schleicher amp Schuell PVDF Millipore Immobilon P neutral nylon Pall Biodyne A or positively charged nylon Pall Biodyne B Bio Rad Zetabind membrane Electroblotting apparatus EC Apparatus Bio Rad or Amersham Pharmacia Biotech Indelible pen e g Paper Mate ballpoint or soft lead pencil Additional reagents and equipment for gel electrophoresis UNIT 6 1 and staining proteins in gels and on membranes see Support Protocol 1 NOTE Deionized distilled water should be used throughout this protocol Contributed by Sean Gallagher Scott E Winston Steven A Fuller and John G R Hurrell Current Protocols in Cell Biology 1998 6 2 1 6 2 20 Copyright 1998 by John Wiley amp Sons Inc UNIT 6 2 BASIC PROTOCOL 1 Electrophoresis and Immunoblotting 6 2 1 Immunoblotting and Immunodetection 6 2 2 Electrophorese the protein sample 1 Prepare antigenic samples and separate proteins using small or standard sized one or two dimensional gels UNIT 6 1 Include prestained or biotinylated protein molecu lar weight standards in one or more gel lanes The protein markers will transfer to the membrane and conveniently indicate membrane orientation and sizes of proteins after immunostaining A variety of gel sizes and percenta
181. er neutral pH nondenaturing conditions The samples and standard proteins should be used at concentrations of 1 to 2 ug l Materials 4x acetic acid gel buffer 200 mM acetic acid pH 3 7 to 5 6 see recipe 4x phosphate gel buffer 400 mM sodium phosphate pH 5 8 to 8 0 see recipe 4x Tris gel buffer 200 mM Tris Cl pH 7 1 to 8 9 see recipe 4x glycine gel buffer 200 mM glycine pH 8 6 to 10 6 see recipe 300 mM sodium sulfite 0 38 g in 10 ml H O used in acetic acid gel preparation Protein samples to be analyzed Contributed by Sean R Gallagher Current Protocols in Cell Biology 2000 6 5 1 6 5 11 Copyright 2000 by John Wiley amp Sons Inc UNIT 6 5 BASIC PROTOCOL Electrophoresis and Immunoblotting 6 5 1 Supplement 5 One Dimensional Electrophoresis Using Nondenaturing Conditions 6 5 2 Supplement 5 Native protein standards Electrophoresis buffer appropriate 4x gel buffer diluted to 1x with H O 75 ml side arm flask used in gel preparation Additional reagents and equipment for gel electrophoresis U7 6 1 and staining proteins in gels 4PPENDIX 3 Prepare the gel 1 Assemble the glass plate sandwich of the gel electrophoresis unit and secure it to the casting stand Either single concentration or gradient gels can be used in the minigel or standard size format Gradient gels will enhance the band sharpness of the separated proteins 2 Prepare acrylamide solutions according
182. er software Amer sham Pharmacia Biotech Clustering was performed using the Dice coefficient with a tree structure based on the Unweighted Pair Group Method using Arithmetic Averages UPGMA Similarity values between isolates can be determined by locating the node that connects the isolates and reading the value from the scale on the lower left edge of the dendrogram Digital Electrophoresis Analysis 6 9 12 Supplement 16 for error the area within a small radius is searched extending from the end of the vector Once another match is found its vector is com puted and used as the starting point for finding neighboring matches From this progression the entire gel is matched If all vectors are displayed graphically when matching is com plete questionable matches can often be iden tified as vectors that are significantly different from neighboring vectors One specialized use of matching is as an estimator of the similarity and potential genetic relatedness of organisms For example on a one dimensional gel image a ratio of the matched to unmatched bands for each pairwise combination of lanes can be calculated This ratio can be used as an indicator of similarity with values near 1 indicating a pair of highly similar lanes and values near zero indicating very dissimilar lanes Assuming that the con tents of the lanes are valid samples of the originating organism s genetic makeup the in formation on lane similari
183. ere the hydrophilic side of a 12 4 x 25 8 cm GelBond film Express any trapped air or excess water with a lint free tissue Mate the glass plate with the adherent GelBond film and the spacer plate Clamp with two flexiclamps The gel forming sandwich should consist of the glass plate the larger of the two plates with an adherent piece of GelBond film hydrophilic side to the glass plate and the spacer plate with its adherent spacer bars placed on top of the GelBond Fig 6 7 1 Place the clamped plates and a 20 ml syringe in a 60 C oven for 10 minutes to equilibrate Measure 40 ml agarose gel buffer into a 50 ml Erlenmeyer flask and add a Teflon coated magnetic stir bar Pour 0 54 g SeaKem HGT P agarose into the flask tightly cover with aluminum foil and place the flask into the boiling water bath step 5 Dissolve the agarose with constant stirring After the solution becomes clear continue boiling an additional 10 min Remove the heated glass plate assembly gel forming sandwich and the 20 ml syringe from the oven Quickly fill the heated syringe with 20 ml hot agarose and holding the plate assembly at an 75 angle fill the narrow space in the plate assembly using a back and forth motion to prevent bubbles from being trapped in the gel Use of a needle with the syringe is optional Current Protocols in Cell Biology Electrophoresis and Immunoblotting 6 7 5 Supplement 15 Agarose Gel Electropho
184. erence Coomassie blue staining has been widely used to visualize protein bands destined for in gel generation of peptides for microse quencing mainly because these downstream applications have not been sensitive enough to allow analysis of protein bands below the de tection limit of the dye Recent methodological improvements however have made it possible to obtain sequence information from as little as 5 ng of protein from polyacrylamide gels stained with silver Wilm et al 1996 Troubleshooting Tables 6 6 1 to 6 6 4 summarize common problems that may arise during staining of proteins in polyacrylamide gels Before using any of the methods to analyze a real sample Current Protocols in Cell Biology it is advisable to test the method first on a gel containing known amounts of standard proteins e g see Fig 6 6 1 Anticipated Results The protocols described in this unit should allow detection of submicrogram amounts of protein separated by PAGE as exemplified by Figure 6 6 1 Time Considerations By far negative staining with zinc is the fastest method among the ones described in this unit Images can be obtained within 20 min after the polyacrylamide gel is removed from the electrophoresis unit Silver staining re quires 30 min for fixation and another 2 5 hr to complete the procedure Both the Coomassie blue and SYPRO Ruby procedures require 3 5 hr for obtaining optimal protein to background staining ratio
185. erest e g UNITS 6 4 amp 15 4 20 w v TCA solution in water IEF Coomassie blue stock solution see recipe 10 w v CuSO in distilled water store for up to 4 months at room temperature CAUTION TCA is extremely caustic Protect eyes and wear gloves when preparing and handling TCA solutions Current Protocols in Cell Biology 1 Make fresh staining solution by mixing 90 ml of destaining solution 10 ml of IEF Coomassie blue stock solution and 1 ml of 10 w v CuSO in distilled water 2 Remove polyacrylamide gel from isoelectric focusing assembly and place it in a plastic container with lid containing 5 to 10 gel vol of 20 w v TCA solution in water 3 Incubate for 30 to 60 min with gentle agitation The use of a platform shaker is recommended In this step proteins are selectively fixed while ampholytes are removed from the gel 4 Remove the TCA solution and add 10 gel vol destaining solution Do not reuse the TCA solution dispose of it following applicable safety regulations for chemical waste In this step the TCA is removed from the gel 5 Incubate 15 min with gentle agitation 6 Discard the destaining solution and add freshly prepared staining solution from step 1 7 Incubate with gentle agitation for 220 min for a gel lt 1 mm thick or 21 hr for a gel gt 1 mm thick 8 Remove the staining solution and rinse the gel briefly with distilled water Do not reuse the staining solution 9 Destain the
186. eries will experience 10 mA The voltage however is additive for each gel If one gel at a constant 10 mA produces 100 V then two identical gels in series will produce 200 V 100 V each and so on Thus the voltage can limit the number of units connected in series on low voltage power supplies Gel thickness affects the above relationships A 1 5 mm gel can be thought of as two 0 75 mm_ thick gels run in parallel Because currents are additive in parallel circuits a 0 75 mm gel will require half the current of the 1 5 mm gel to achieve the same starting voltage and separation time If gel thickness is doubled then the current must also be doubled There are limits to the amount of current that can be applied Thicker gels require more current generating more heat that must be dissipated Unless temperature control is available in the gel unit a thick gel should be run more slowly than a thin gel NOTE Milli Q purified water or equivalent should be used throughout the protocols DENATURING SDS DISCONTINUOUS GEL ELECTROPHORESIS LAEMMLI GEL METHOD One dimensional gel electrophoresis under denaturing conditions i e in the presence of 0 1 SDS separates proteins based on molecular size as they move through a polyacryl amide gel matrix toward the anode The polyacrylamide gel is cast as a separating gel sometimes called resolving or running gel topped by a stacking gel and secured in an electrophoresis apparatus After sample proteins ar
187. es are either horizontal or vertical Since band and spot detection will be much easier if the image is properly oriented this eliminates the need to later rotate the image digitally Image rotation is time consuming and can result in spatial linearity errors a change in the size and shape of objects in image caused by rectangular image capture device geome tries The next step for camera based systems is to adjust magnification and to focus the sample image For thicker samples it might be necessary to reduce the aperture on camera based systems to get a sufficient depth of field to focus the entire sample Often at this point image imperfections e g dust liquid or other foreign objects that will detract from later analysis are detected and they need to be removed Next image intensity is set Within the area of interest on the image band or spot peaks should have values less than the maxi mum saturated value and the background should have nonzero values This is usually accomplished through adjustment of the light source intensity or the sensor signal integration If the device allows precapture optimization of other parameters such as spatial resolution contrast brightness gain or gamma they are adjusted next Note that this only applies to controls that affect the response of the sensor or processing of the image prior to a data reduction step and not to controls that affect the image at later stages The latter pr
188. es to polymerize at different heights 10 Drain off the overlay and rinse the surface of the gels with 1 x Tris Cl SDS pH 8 8 Pour stacking gel and remove gels from caster 11 Prepare and cast the stacking gel as in casting multiple single concentration gels see Support Protocol 1 steps 8 to 11 12 Remove gels from the caster and clean the gel sandwiches see Support Protocol 1 steps 12 and 13 Store gels if necessary according to the instructions for multiple single concentration gels see Support Protocol 1 step 14 ELECTROPHORESIS IN SINGLE CONCENTRATION MINIGELS Separation of proteins in a small gel format is becoming increasingly popular for ap plications that range from isolating material for peptide sequencing to performing rou tine protein separations The unique combination of speed and high resolution is the foremost advantage of small gels Additionally small gels are easily adapted to single concentration gradient and two dimensional SDS PAGE procedures The minigel pro cedures described are adaptations of larger gel systems This protocol describes the use of a multiple gel caster The caster is simple to use and up to five gels can be prepared at one time with this procedure Single gels can be prepared using adaptations in the manufacturer s instructions A multiple gel caster is the only practical way to produce small linear polyacrylamide gradient gels see Support Protocol 3 Materials Minigel ver
189. escence the gel can be washed in a mixture of 10 methanol or ethanol and 7 acetic acid for 30 min instead of water The gel may be monitored periodically using UV illumination to determine the level of background fluorescence 15 Visualize the stain using an appropriate method see Fig 6 8 1 The stained gel is best viewed on a standard 300 nm UV B transilluminator though stain will be visible using a 254 nm UV C or 365 nm UV A transilluminator Gels may also be visualized using various laser scanners 473 nm SHG laser 488 nm argon ion laser or 532 nm YAG laser Alternatively use a xenon arc lamp blue fluorescent light or blue light emitting diode LED source Gels may be photographed by Polaroid or CCD camera Use Polaroid 667 black and white print film and the SYPRO protein gel stain photographic filter Molecular Probes FLUORESCENT DETECTION OF GLYCOPROTEINS ON ELECTROBLOT MEMBRANES Pro Q Emerald 300 Glycoprotein Blot Stain Kit provides a robust method for differen tially staining glycosylated and non glycosylated proteins on the same electroblot The technique combines the green fluorescent Pro Q Emerald 300 glycoprotein stain with the orange red fluorescent SYPRO Ruby total protein gel stain The Pro Q Emerald 300 glycoprotein stain reacts with periodate oxidized carbohydrate groups creating a bright green fluorescent signal on glycoproteins Using this stain allows detection of lt 1 ng glycoprotein band depending u
190. esired to speed polymerization degas under vacuum 5 min Add 10 ammonium persulfate and TEMED Swirl gently to mix Use immediately The recipes produce 40 ml gel solution which is adequate for one gel of dimensions 1 5 mm x 14 cm x 16 cm or two gels of dimensions 0 75 mm x 14 cm x 16 cm The pH range can be extended to 2 0 the pH of acetic acid by using unadjusted acetic acid in place of 4x acetic acid gel buffer although there is little buffering capacity at this pH All reagents and solutions used in the protocol must be prepared with Milli Q purified water or equivalent 4Units of numbers in table body are milliliters The desired percentage of acrylamide in the gel solution depends on the molecular size of the protein being separated Must be freshly made Sodium sulfite is needed for efficient polymerization at acid pH SAdded just before polymerization Current Protocols in Cell Biology Table 6 5 2 Recipes for Phosphate Nondenaturing Polyacrylamide Gels pH Range 5 8 to 8 0 Final acrylamide concentration in gel 5 7 5 10 12 5 15 17 5 20 Stock solution 30 acrylamide 0 8 6 7 10 13 3 16 8 20 23 32 26 6 bisacrylamide 4x phosphate gel buffer 10 10 10 10 10 10 10 H 0 23 08 19 78 1648 12 98 9 78 646 3 18 10 w v ammonium 0 2 0 2 0 2 0 2 0 2 0 2 0 2 persulfate TEMED 0 02 0 02 0 02 0 02 0 02 0 02 0 02 Preparation of gel In a 75 ml side arm flask mix 30 acrylamide 0 8 bisa
191. esolutions more bands are detectable the maximum end of the dynamic range This results in a loss of detail and quantitative infor mation from those data points that are saturated For fluorescent and luminescent samples re duction in the sampling time can sometimes correct saturation problems Optical density based visualization techniques can also gener ate saturated images as is illustrated in Figure 6 9 1E this can sometimes be avoided with longer sampling times or increased detection source intensities More often it will be neces sary to perform another electrophoresis with more dilute samples or to alter the visualization process to generate a less optically dense ma terial Resolution Resolution is the ability of a system to dis tinguish between two closely placed or similar objects Three types of resolution are important for analysis spatial resolution intensity reso lution and technique dependent resolution Spatial resolution is the ability to detect two closely placed objects in one two or three dimensional space It is most accurately de scribed as the closest distance two objects can be placed and still be detected as separate ob jects In practice it is often defined nominally in terms of the number of detectors per unit area such as dots per inch dpi or the number of detectors present in total or in each dimension such as 512 x 512 262 144 total detectors Actual resolution is less than half
192. et acrylamide solutions by mouth Agarose in reducing buffer 1 5 w v Mix 0 15 g agarose and 10 ml reducing buffer see recipe Heat in boiling water bath until dissolved Prepare immediately before use Agarose 2 w v Mix 2 g agarose and 100 ml water Stir on a hot plate until dissolved Keeping the solution near 100 C divide by placing 5 ml aliquots in 25 ml glass screw cap tubes Let the aliquots solidify Store at 4 C stable at least 3 months Ammonium persulfate 2 5 w v 0 25 g ammonium persulfate H O to 10 ml Prepare immediately before use DNase and RNase solution Mix 2 50 ml of 1 M Tris Cl pH 7 0 APPENDIX 24 250 ul of 1 M MgCl APPENDIX 24 and 2 2 ml water Add to 5 mg DNase Worthington and dissolve DNase Add 2 5 mg RNase in solution Worthington Mix well divide into 50 or 100 ul aliquots and store at 80 C stable at least 1 year The volume of RNase solution needed is variable and depends on the protein concentration which is reported on the vial label in mg ml The volume of RNase added should be lt 200 ul if a larger volume is used the amount of H O should be decreased proportionally Final concentrations are 0 5 M Tris Cl pH 7 0 0 1 M MgCl 0 1 w v DNase and 0 05 RNase DryStrip equilibration solutions 20 ml 1 M Tris Cl pH 6 8 APPENDIX 24 72 g ultrapure urea 60 ml glycerol 2 g sodium dodecyl sulfate SDS 67 ml Milli Q purified water For solution 1 Add 50 mg DTT per 10 ml
193. etected in a de fined complex but not in a reproducible man ner In this case the complex might have been an artifact generated during the electrophore sis step This could be due to a step in the gel gradient i e the gradient was not continu ous When pouring the gradient make sure that there is a continuous flow of liquid and also that the flow is continuous between the two cylinders of the gradient maker While loading the first dimension gel slice onto the second dimension SDS PAGE there could have been a small air bubble under the slice This pre vents entry of protein along that vertical line giving the impression that two MPCs exist rather than one If a certain interaction between two pro teins is not seen by co immunopurifications do not expect to detect it by BN PAGE If the protein of interest is only found as a monomer although it should be present in a complex the complex could have been dis rupted by detergent or be of low abundance Thus the choice of detergent is important in extracting but not disrupting multiprotein complexes Without detergent proteins tend to aggregate during the stacking step of the elec trophoresis and do not enter the separating gel properly Thus a certain concentration of de tergent has to be present in the sample Unfor tunately general rules for the choice of deter gent do not exist Compare several detergents of different classes and three different concen trations of e
194. f 0 2 um membranes may improve retention of smaller molecular weight pro teins 8 Wet another piece of Whatman 3MM filter paper place on anode side of membrane and remove all air bubbles Place another Scotch Brite pad or sponge on top of this filter paper 9 Complete assembly by locking top half of the transfer cassette into place Fig 6 2 1 It is important to orient the sandwich so that the membrane faces the anode positively charged side of the tank Transfer proteins from gel to membrane 10 Fill tank with transfer buffer and place transfer cassette containing sandwich into electroblotting apparatus in correct orientation Connect leads of power supply to corresponding anode and cathode sides of electroblotting apparatus Transfer buffer should cover the electrode panels but should not touch the base of the banana plug 11 Electrophoretically transfer proteins from gel to membrane for 30 min to 1 hr at 100 V with cooling or overnight at 14 V constant voltage in a cold room Transfer time is dependent on the thickness and the percent acrylamide of the gel as well as the size of the protein being transferred In general proteins are transferred within 1 to 6 hr but high molecular weight molecules may take longer Overnight transfer at low voltage is reliable and convenient Cooling at 10 to 20 C is required for transfers gt 1 hr 7 j Electrophoresis at high power Heat exchanger cooling cores using a c
195. f very acidic Alternate Protocol 1 or very basic Alternate Protocol 2 proteins In both cases prefocusing of the gels must be avoided and the isoelectro focusing time has to be reduced A short separa tion time does not allow the system to reach equilibrium and is used to establish the desired Electrophoresis and Immunoblotting 6 4 31 Supplement 4 Table 6 4 3 Commercially Available Precast IPG Gels and Immobiline Chemicals Name Use Available pH range Immobiline DryPlate Running one dimensional immobilized pH 4 0 7 0 pH 4 2 4 9 pH 4 5 5 4 pH gradient gels pH 5 0 6 0 pH 5 6 6 6 Immobiline DryStrip Running first dimension in two dimensional gels 110 mm pH 4 7L pH 3 10L 180 mm pH 4 7L pH 3 10L pH 3 10NL IPG Ready Strips Running first dimension in two dimensional gels 7 cm pH 3 10 pH 4 7 pH 3 6 pH 5 8 pH 7 10 11 cm pH 3 10 pH 4 7 pH 3 6 pH 5 8 pH 7 10 Immobiline II Creating custom gradient immobilized pHgradient gels pK 3 6 pK 4 6 pK 6 2 pK 7 0 pK 8 5 pK 9 3 From Amersham Pharmacia Biotech gt From Bio Rad CA linear gradient with maximum resolution above pH 7 0 dA nonlinear gradient with best resolution at pH 5 0 7 0 Two Dimensional Gel Electrophoresis 6 4 32 Supplement 4 pH gradient Separation of very acidic proteins requires modifications of gel and electrode so lutions Separation of very basic proteins has to be performed with the posi
196. factor with vWF and this immobilization step was eliminated from the method Electrophoresis and Immunoblotting 6 7 11 Supplement 15 Agarose Gel Electrophoresis of Proteins 6 7 12 Supplement 15 900 kDa p 700 kDa gt 204 kDa 1 6 14 a o 5 l Optical density oO pit PNP o oO 2 P l De O 10 20 30 40 50 60 70 80 90 Millimeters Figure 6 7 3 Luminograph of vWF multimers in pooled normal plasma PNP lanes 1 and 3 and vWF multimers extracted from platelets plt newly synthesized vWF lanes 2 and 4 The densi tometric tracing below shows the earlier take off of the platelet vWF indicating larger multimers Adapted with permission from Krizek and Rick 2000 Analysis of the distribution of von Wille brand factor multimers is also used to assess the function of an important protease that cleaves von Willebrand factor and decreases the prothrombotic unusually high molecular weight multimers of von Willebrand factor these multimers are initially synthesized and secreted into the circulation but are cleaved by the vWF protease Krizek and Rick 2001 Aronson Krizek and Rick 2001 Critical Parameters and Troubleshooting It is important to maintain the temperature at 4 C during electrophoresis using the hori zontal bed see Basic Protocol The blotting step must be carried out for a sufficient time to a
197. fan ner for initially introducing me to BN PAGE and Hermann Schagger for his invaluable help in setting up the technique I further thank Balbino Alarc n for his advice on establishing the NAMOS assay and mem bers of my laboratory for constantly improv ing the various BN techniques Margarita Camacho Carvajal Mahima Swamy Thomas Bock Eszter Molnar Susana Minguet Elaine Pashupati Dopfer and Gabrielle Siegers Current Protocols in Cell Biology I also acknowledge financial support by the European Union through an individual Marie Curie fellowship and the Deutsche Forschungsgemeinschaft through the Emmy Noether program SCHA 976 1 and the SFB620 Literature Cited Camacho Carvajal M M Wollscheid B Aebersold R Steimle V and Schamel W W 2004 Two dimensional blue native SDS gel electrophoresis of multi protein complexes from whole cellular lysates A proteomics approach Mol Cell Proteomics 3 176 182 Dudkina N V Eubel H Keegstra W Boekema E J and Braun H P 2005 Structure of a mito chondrial supercomplex formed by respiratory chain complexes I and III Proc Natl Acad Sci U S A 102 3225 3229 Millar A H Heazlewood J L Kristensen B K Braun H P and Moller I M 2005 The plant mitochondrial proteome Trends Plant Sci 10 36 43 Model K Meisinger C Prinz T Wiedemann N Truscott K N Pfanner N and Ryan M T 2001 Multistep assembly of the protein import channel
198. few minutes to a few weeks depending on the strength of the radioactivity in the sample Most exposures last from several hours to a few days Exposure time can be reduced more than 10 fold with a phos phor imager Literature Cited Chamberlain J P 1979 Fluorographic detection of radioactivity in polyacrylamide gels with the water soluble fluor sodium salicylate Anal Biochem 98 132 135 Johnston R F Pickett S C and Barker D L 1990 Autoradiography using storage phosphor tech nology Electrophoresis 11 355 360 Laskey R A 1980 The use of intensifying screens or organic scintillators for visualizing radioac tive molecules resolved by gel electrophoresis Methods Enzymol 65 363 371 Laskey R A and Mills A D 1975 Quantitative film detection of H and C in polyacrylamide gels by fluorography Eur J Biochem 56 335 341 Laskey R A and Mills A D 1977 Enhanced auto radiographic detection of P and I using in tensifying screens and hypersensitized film FEBS Lett 82 314 316 Contributed by Daniel Voytas and Ning Ke Iowa State University Ames Iowa Current Protocols in Cell Biology Two Dimensional Gel Electrophoresis Two dimensional gel electrophoresis combines two different electrophoretic separating techniques in perpendicular directions to provide a much greater separation of complex protein mixtures than either of the individual procedures The most common two dimen sional techni
199. ffer should contain SDS 5 After transfer soak membranes for 10 to 30 min in 45 methanol nitrocellulose or 100 methanol nylon or PVDF to remove the bound Coomassie blue This step is not needed if using chemiluminescent reactions or radiolabeled protein A for immunodevelopment Destaining of the nitrocellulose membrane is enhanced by adding a small ball of laboratory tissue to the methanol to absorb the Coomassie blue 6 Proceed with immunoprobing and visual detection of proteins see Basic Protocols 2 and 3 and Alternate Protocols 3 and 4 REVERSIBLE STAINING OF TRANSFERRED PROTEINS To verify transfer efficiency nitrocellulose and PVDF membranes can be reversibly stained This method will not work on nylon membranes Additional Materials also see Basic Protocol 1 Ponceau S solution see recipe Additional reagents and equipment for photographing membranes 1 Following protein transfer to nitrocellulose or PVDF see Basic Protocol 1 or Alternate Protocol 1 place membrane in Ponceau S solution 5 min at room temperature 2 Destain 2 min in water Photograph membrane if required and mark the molecular weight standard band locations with indelible ink 3 Completely destain membrane by soaking an additional 10 min in water QUANTITATION OF PROTEIN WITH PONCEAU S In addition to qualitatively visualizing proteins on membranes after blotting Ponceau S provides a convenient method for quantifying the amount of protein i
200. fficiently than thicker gels e g 1 5 mm thick Gels with a higher acryl amide percentage will also transfer less effi ciently Proteins can be particularly difficult to transfer from gradient gels and a combination of longer transfer times thin gels and the addition of SDS to the transfer buffer may be needed If the protein bands are diffuse check the transfer cassette The gel must be held firmly against the membrane during transfer If the transfer sandwich is loose in the cassette add another thin sponge or more blotter paper to both sides Occasionally a grid pattern will be apparent on the membrane after tank transfer This is caused by having either the gel or the membrane too close to the sides of the cassette Correct this by adding more layers of filter paper to diffuse the current flowing through the gel and membrane Use a thinner sponge and more filter paper if necessary If air bubbles are trapped between the filter and the gel they will appear as clear white areas on the filter after blotting and staining Take extra care to make sure that all bubbles are removed Insufficient blocking or nonspecific binding of the primary or secondary antibody will cause a high background stain A control using pre immune serum or only the secondary antibody will determine if these problems are due to the primary antibody Try switching to another blocking agent protein blocking agents may weakly cross react Lowering the co
201. film the highest sensitivity is achieved with a 490 nm long pass filter such as the SYPRO protein gel stain photographic filter S 6656 Molecular Probes Gels are typically photo graphed using an f stop of 4 5 for 2 to 4 sec using multiple 1 sec exposures Using a CCD camera images are best obtained by digitizing at 1024 x 1024 pixels resolution with 12 14 or 16 bit gray scale levels per pixel A 520 nm long pass filter is suitable for visualiz ing Pro Q Emerald dye while a 580 nm long pass filter is appropriate for detection of DDAO A CCD camera based image analysis system can gather quantitative information that will allow comparison of fluorescence intensi ties between different bands or spots Using such a system the Pro Q Emerald dye and the DDAO dye have a linear dynamic range of three orders of magnitude A potential problem associated with the Pro Q Emerald 300 glycoprotein gel stain is non specific labeling of non glycosylated proteins The most common source of this problem is the presence of residual SDS in the polyacrylamide gel Adding an extra fixation step to the proce dure should prevent its occurrence The author finds that an overnight fixation step for two di mensional gels is advisable When detecting glycans using Pro Q Emerald 300 glycoprotein gel stain it is prudent to run control gels in which the periodate oxidation step has been omitted Similar precautions are advisable when evaluating result
202. film has been properly preexposed see Support Protocol 3 The linear range of correctly preexposed film is 0 1 to 1 0 absorbance units However if the preexposure is excessive i e an increase of gt 0 2 absorbance units A549 treated film untreated film smaller amounts of radioactivity will produce disproportionately dense images Autoradiograms that exceed an absorbance of 1 4 absorbance units A549 have saturated all available silver bromide crystals and also cannot be evaluated quantitatively Densitometers are available from several manufacturers e g Molecular Dynamics Bio Rad and UVP Most models come with software that facilitates calculations and allows the user to define the region of the film to be measured Procedures for the use of these machines vary and instructions are provided by the manufacturer Densitometers are also available that measure light reflected from a sample Reflectance densitometers are useful in instances where the sample medium is completely opaque e g filters that have been probed using nonradioactive colorimetric detection assays Current Protocols in Cell Biology ALTERNATE PROTOCOL 1 SUPPORT PROTOCOL 4 Electrophoresis and Immunoblotting 6 3 7 ALTERNATE PROTOCOL 2 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots 6 3 8 PHOSPHOR IMAGING Phosphor imaging screens can be used as an alternative to film for recording and quantifying autoradi
203. flows in from the bottom To use a multiple casting unit the light solution is placed in the mixing chamber and the heavy solution in the reservoir This is the reverse of casting a single gel see Alternate Protocol 5 Thus light solution enters the multiple caster first followed by progressively heavier solution Finally the acrylamide solution is stabilized in the multiple caster with a heavy plug solution and allowed to polymerize see step 8 and manufacturer s instructions 7 When almost all the acrylamide solution is gone from the gradient maker stop the pump and close the mixing valve Tilt the gradient maker toward the outlet side and remove the last milliliters of the mix Do not allow air bubbles to enter the tubing 8 Add the plug solution to the mixing chamber and start the pump Make sure that no bubbles are introduced Continue pumping until the bottom of the caster is filled Electrophoresis with plug solution to just below the glass plates then turn off the pump Clamp the Doaanobioitin tubing close to the red port of the casting chamber 6 1 23 Current Protocols in Cell Biology Supplement 37 BASIC PROTOCOL 2 One Dimensional SDS PAGE 6 1 24 Supplement 37 9 Quickly overlay each separate gel sandwich with 100 pl H2O saturated isobutyl alcohol Use the same amount on each sandwich Allow the gels to polymerize for Thr Failure to use the same amount of overlay solution will cause the gel sandwich
204. for process monitoring and the quality assessment of recombinant glycoproteins Electrophoresis 19 2572 2594 Key References Steinberg et al 2001 See above Describes detection of glycoproteins in gels and on blots using Pro Q Emerald 300 Glycoprotein Detec tion Kits as well as the detection of concanavalin A binding and wheat germ agglutinin binding gly coproteins on blots using lectin alkaline phos phatase conjugates and DDAO phosphate Current Protocols in Cell Biology Berggren K Steinberg T Lauber W Carroll J Lopez M Chernokalskaya E Zieske L Diwu Z Haugland R and Patton W 1999 A luminescent ruthenium complex for ultrasensi tive detection of proteins immobilized on mem brane supports Anal Biochem 276 129 143 Describes counter staining with SYPRO Ruby dye for the detection of total protein profiles on electrob lot membranes Berggren K Chernokalskaya E Steinberg T Kemper C Lopez M Diwu Z Haugland R and Patton W 2000 Background free high sensitivity staining of proteins in one and two dimensional sodium dodecyl sulfate polyacry lamide gels using a luminescent ruthenium com plex Electrophoresis 21 2509 2521 Describes counter staining with SYPRO Ruby dye for the detection of total protein profiles in polyacry lamide gels Lopez M Berggren K Chernokalskaya E Laz arev A Robinson M and Patton W 2000 A comparison of silver stain an
205. for making acrylamide solutions casting separating gels stacking gels are omitted loading samples and conducting electrophoresis Continuous systems although flexible do not give the high resolution separation found in discontinuous systems see Alternate Protocol Separation in a continuous system i e in which the same buffer is used for preparing acrylamide solutions and filling electrophoresis chambers is governed by pH and this protocol describes four types of buffers useful over discrete ranges from pH 3 7 to pH 10 6 Use of unadjusted acetic acid gel buffer can extend the range to pH 2 0 The choice of pH and thus the buffer system depends on the protein being studied i e its isoelectric point and often must be determined empirically In general the system should be between pH 5 0 and 8 0 for optimal results Extremes of pH can lead to precipitation or denaturation of the protein Acrylamide concentrations are empirically determined but the higher the percent acrylamide the sharper the protein bands It is important to include native protein standards in the electrophoresis runs Several manufacturers supply standards for isoelectric focusing that are also suitable for native electrophoresis The standards have a range of isoelectric points and will carry a net positive negative or zero charge depending on the pH of the gel system Alternatively Sigma supplies a standard kit that is useful for calculating molecular weights und
206. fresh before use CAUTION Formaldehyde is toxic and carcinogenic Wear gloves and handle the concen trated 37 solution in a chemical hood Fixative solution Mix 50 ml of methanol with 12 ml glacial acetic acid and add water to 100 ml Add 50 ul of 37 formaldehyde Prepare fresh before use CAUTION Glacial acetic acid and methanol are volatile and toxic Formaldehyde is toxic and carcinogenic Wear gloves and prepare the solution in a chemical fume hood Current Protocols in Cell Biology Electrophoresis and Immunoblotting 6 6 7 Supplement 6 Staining Proteins in Gels 6 6 8 Supplement 6 IEF Coomassie blue stock solution Dissolve 0 2 w v Coomassie brilliant blue R 250 and 60 v v methanol in water Store for up to 4 months at room temperature Silver nitrate solution Dissolve 0 2 g AgNO in 100 ml water Add 75 ul of 37 formaldehyde Protect the solution from light Prepare fresh before use CAUTION Formaldehyde is toxic and carcinogenic Wear gloves and handle the concen trated 37 solution in a chemical fume hood Discard the solution if it becomes cloudy Thiosulfate solution Dissolve 20 mg Na SO 5H O in 100 ml water Prepare fresh before use COMMENTARY Background Information The first protocols developed for the visu alization of proteins bands after electrophore sis in polyacrylamide or other gel matrices relied on the use of dyes with selective avidity for polypept
207. g chamber making sure the stack fits snugly Secure the plate with four spring clamps and tighten the bottom thumb screws 4 Prepare the separating resolving gel solution Table 6 1 9 A 12 cm separating gel with a 4 cm stacking gel is recommended lf fewer than ten gels are prepared Table 6 1 9 use the following formula to estimate the amount of separating gel volume needed Electrophoresis an volume gel no x height cm x width cm x thickness cm 4 x gel no 10 ml Immunoblotting 6 1 17 Current Protocols in Cell Biology Supplement 37 ALTERNATE PROTOCOL 5 One Dimensional SDS PAGE 6 1 18 Supplement 37 5 Using a 100 ml disposable syringe with flat tipped needle inject the resolving gel solution down the side of one spacer into the multiple caster A channel in the silicone plug distributes the solution throughout the whole caster Avoid introducing bubbles by giving the caster a quick tap on the benchtop once the caster is filled 6 Overlay the center of each gel with 100 u1 H2O saturated isobutyl alcohol and let polymerize for 1 to 2 hr 7 Drain off the overlay and rinse the surface with 1 x Tris Cl SDS pH 8 8 If the gels will not be used immediately skip to step 12 Pour the stacking gel 8 Prepare the stacking gel solution either singly see Basic Protocol 1 step 8 or for all the gels at once Table 6 1 9 The stacking gel solution should be prepared just before it is poured 9 Fill
208. g gel spacers and combs can be cut to fit 1 Wash gel plates with water based laboratory detergent followed by successive rinses with hot tap water deionized water and finally 95 ethanol Allow to air dry Gel plates must be extremely clean for casting thin gels Gloves are needed throughout these procedures to prevent contamination by proteins on the surface of skin 2 Apply a streak of adhesive from a glue stick to the bottom edge of the glass plate Quickly position the GelBond with the hydrophobic side down a drop of water will bead up on the hydrophobic surface Apply pressure with Kimwipe tissue to attach the GelBond firmly to the plate Finally pull the top portion of the GelBond back place a few drops of water underneath and roll flat with an ink roller Make sure the GelBond does not extend beyond the edges of the upper and lower sealing surface of the plate This will cause it to buckle on sealing Reposition the GelBond if needed to prevent it from extending beyond the glass plate Material may also be trimmed to fit flush with the plate edge Current Protocols in Cell Biology ALTERNATE PROTOCOL 4 Electrophoresis and Immunoblotting 6 1 15 Supplement 37 SUPPORT PROTOCOL 1 One Dimensional SDS PAGE 6 1 16 Supplement 37 Assemble the gel cassette according to the manufacturer s instructions also see Basic Protocol 1 steps 1 and 2 Just prior to assembly blow air over the surface of both
209. g more time consuming two dimensional gels prescreening fractions from a chromatographic purification step prior to running two di mensional gels and evaluation of charge heterogeneity of purified proteins Additional Materials also see Basic Protocol 2 Precast DryPlate gel Amersham Pharmacia Biotech Repel Silane Amersham Pharmacia Biotech Paraffin oil Protein samples to be analyzed Reswelling Cassette kit Amersham Pharmacia Biotech including 125 x 260 x 3 mm glass plate with 0 5 mm U frame 125 x 260 x 3 mm glass plate Silicone tubing Pinchcock Clamps 20 ml syringe Roller Amersham Pharmacia Biotech Whatman no 1 filter paper Flatbed electrophoresis unit Amersham Pharmacia Biotech Multiphor IT 10 or 15 C cooling water bath Electrode strips Sample applicator strip or sample application pieces Power supply minimum capacity 3000 to 3500 V Additional reagents and equipment for protein detection by staining APPENDIX 3 and for electroblotting UNIT 6 2 optional Rehydrate the gel 1 Remove precast gel from packaging If the entire gel is not needed cut off the required number of lanes and reseal the unused gel Mark the polarity of the gel section to be used by cutting a small triangle off the anode corner Handle the gel by the support film only It is critical that the lanes are cut from the gel in the proper orientation to preserve the pH gradient see Fig 6 4 1 and polarity must be indicated for proper o
210. gel the ends of the gel strip may be trimmed away from the IPG gel so that it will fit on top of the second dimension however some very basic or acidic proteins may be lost 2 Pour a separating gel of the desired acrylamide concentration and immediately overlay with water to produce a smooth surface The separating gel should be a minimum of 2 5 cm below the top of the inner plate to accommodate a 2 cm stacking gel 3 After the separating gel has polymerized remove the water overlay rinse the gel surface with water to remove any unpolymerized acrylamide and pour the stacking gel to a height of 0 5 cm from the top of the plate Overlay with water to produce a smooth surface A water overlay provides a smooth surface for better contact between the Immobiline DryStrip and the second dimension gel The stacking gel height should be 2 cm Load the Immobiline IPG DryStrip gel onto the second dimension gel 4 Prepare Immobiline DryStrip equilibration solutions and 2 see recipe 5 Assemble the second dimension gels in a electrophoresis chamber Do not pour electrophoresis buffer into the upper chamber 6 Melt 2 w v agarose in a boiling water bath Mix a solution of 1 part 2 agarose to 2 parts equilibration solution 2 Keep agarose equilibration buffer mixture in boiling water bath until step 11 is completed The agarose prevents the IPG DryStrip from shifting position and ensures good contact between the IEF and second dimensi
211. gel and should be gently peeled away The gel is now ready to be placed in the strip aligner on the cooling plate of the electrophoresis unit Do not allow the gel to dehydrate prior to placing it on the cooling plate in step 11 Steps 7 to 10 should be completed prior to removing the strips from the reswelling tray Run the first dimension 7 Level the Multiphor II electrophoresis unit then connect it to a circulating cooling water bath Allow it to cool to 15 C for 1 to 2 hr to ensure even cooling Do not cool below 15 C to prevent precipitation of urea in the gels Pipet 5 ml DryStrip cover fluid onto the surface of the Multiphor II cooling plate Position the Immobiline DryStrip tray on the cooling plate oriented with the red anodic electrode at the top near the cooling tubes Avoid large air bubbles between the cooling plate and the tray small bubbles should not cause a problem Connect the red and black electrode leads on the tray to their respective positions on the Multiphor II unit Pour 10 ml of DryStrip cover fluid into the tray Place the Immobiline DryStrip aligner on top of the oil groove side up Avoid getting oil on top of the strip aligner The possible presence of small air bubbles under the strip aligner is not important Cut two electrode strips to a length of 11 cm regardless of the number of DryStrips used Place the electrode strips onto a clean glass plate and soak each one with 0 5 ml
212. gel solution sufficient for two 0 75 mm gels 7 ml of each concentration or one 1 5 mm gel 14 ml of each concentration Volumes can be adjusted to accommodate gel sandwiches of different dimensions Additional Materials also see Basic Protocol 1 Light and heavy acrylamide gel solutions Table 6 1 10 Bromphenol blue optional for checking practice gradient 10 ammonium persulfate prepare fresh TEMED Current Protocols in Cell Biology Gradient maker 30 to 50 ml Hoefer SG30 or SG50 or 30 to 100 ml Bio Rad 385 Tygon tubing with micropipet tip Peristaltic pump optional e g Markson A 13002 A 34040 or A 34105 minipump Whatman 3MM filter paper Set up the gradient maker and prepare the gel solutions 1 Assemble the magnetic stirrer and gradient maker on a ring stand as shown in Figure 6 1 2 Connect the outlet valve of the gradient maker to Tygon tubing attached to a micropipet tip that is placed over the vertical gel sandwich If desired place a peristaltic pump in line between the gradient maker and the gel sandwich A peristaltic pump will simplify casting by providing a smooth flow rate 2 Place a small stir bar into the mixing chamber of the gradient maker 1 e the chamber connected to the outlet Table 6 1 10 Light and Heavy Acrylamide Gel Solutions for Gradient Gels Acrylamide concentration in light gel solution Stock solution 5 6 7 8 9 10 11 12 13 14 30 acrylamide 0 8 2
213. gentle agitation e g on an orbital shaker at 50 rpm for 45 min at room temperature 4 Wash the gel by incubating it in 50 ml wash solution with gentle agitation for 10 min at room temperature Repeat wash one additional time 5 Oxidize the gel in 25 ml periodic acid solution with gentle agitation for 30 min at room temperature 6 Wash the gel in 50 ml wash solution with gentle agitation for 5 to 10 min at room temperature Repeat this washing step two additional times Stain gel for glycoproteins 7 Prepare fresh Pro Q Emerald 300 staining solution by diluting the 50x Pro Q Emerald 300 concentrate reagent 50 fold into Pro Q Emerald 300 dilution buffer For example dilute 500 ul of 50x Pro Q Emerald 300 reagent into 25 ml dilution buffer to make enough staining solution for one 8 X 10 cm gel 8 Incubate the gel in subdued light in 25 ml of Pro Q Emerald 300 staining solution step 7 while gently agitating for 90 to 120 min The signal can be seen after 20 min and maximum sensitivity is reached at 120 min Staining overnight is not recommended 9 Wash the gel with 50 ml wash solution for 15 min at room temperature Repeat this wash one additional time Do not leave the gel in wash solution for gt 2 hr as the staining signal will start to decrease 10 Visualize the stain using a standard UV transilluminator The Pro Q Emerald 300 stain has an excitation maximum at 280 nm and an emission maximum near 530 nm Stained glycoproteins
214. ges of acrylamide can be used UNIT 6 1 Most routinely used are either 14 cm X 14 cm x 0 75 mm gels or 8 cm X 10 cm Xx 0 75 mm minigels Acrylamide concentrations vary from 5 to 20 but are usually in the 10 to 15 range Assemble the immunoblot sandwich 2 When electrophoresis is complete disassemble gel sandwich and remove stacking gel Equilibrate gel 30 min at room temperature in transfer buffer Oil from hands blocks the transfer Match the appropriate transfer buffer to the membrane see Reagents and Solutions Gel equilibration is required to prevent a change in the size of the gel during transfer Any shift in gel dimension will result in a blurred transfer pattern 3 Assemble transfer sandwich in a tray large enough to hold the plastic transfer cassette Fill with transfer buffer so that cassette is covered The transfer cassette should be assembled under buffer to minimize trapping of air bubbles Use Figure 6 2 1 as a guide to assembly 4 Onbottom half of plastic transfer cassette place Scotch Brite pad or sponge followed by a sheet of filter paper cut to same size as gel and prewet with transfer buffer pad cathode anode plastic support ih filter paper gel nitrocellulose electroblotting buffer ot direction of protein transfer Figure 6 2 1 Immunoblotting with a tank blotting unit The polyacrylamide gel containing the pro
215. ham flash unit 2 Place the film perpendicular to the light source at a distance of 250 cm to ensure uniform illumination 3 Expose a series of test films for different flash lengths then develop them see Basic Protocol An uneven fog level on film can be remedied by placing a porous paper diffuser such as Whatman no I filter paper between the film and the light source 4 Cut the films into pieces that fit into a cuvette holder of a spectrophotometer and measure the absorbance at 540 nm Choose an exposure time that causes the absorbance of the preexposed film to increase by 0 15 with respect to film that was not preexposed Current Protocols in Cell Biology FLUOROGRAPHY Organic scintillants can be included in radioactive samples to obtain autoradiograms of weak f emitting isotopes such as H 14C and S The scintillant fluoresces upon absorption of B particles from these isotopes facilitating film exposure Fluorographs of radioactive molecules in polyacrylamide gels have traditionally used the scintillant PPO 2 5 diphenyloxazole Laskey and Mills 1975 PPO however has largely been replaced with commercial scintillation formulations that reduce the amount of preparation time and are considerably safer to use These scintillants e g Enhance from NEN Life Science come with complete instructions for their use In addition spray applicators are also available that can be used on filters or thin layer plates The expec
216. hance to correct an omission in the procedure especially for samples in limited supply UNIT 6 2 however the immunoblotting step of such large proteins must be done with care in order to assure the adequate transfer of the largest proteins The Alternate Protocol uses direct identification of the protein in the gel by a specific radiolabeled antibody eliminating the immunoblot ting step but sacrificing sensitivity AGAROSE GEL ELECTROPHORESIS AND BLOTTING WITH IMMUNODETECTION The following is a method for separating a complex mixture of human plasma proteins by continuous SDS horizontal submerged agarose gel electrophoresis The very large multimers of circulating plasma von Willebrand factor or more highly purified prepara tions of this protein are identified using a specific antibody and visualized using a chemiluminescent reagent This protocol utilizes a 20 x 25 cm horizontal gel apparatus Dry agarose is weighed mixed with electrophoresis buffer and melted in a hot water bath The agarose is allowed to partially cool then is poured into a horizontal casting frame with a Teflon comb in place and allowed to solidify The gel is covered with 1 to 2 mm cold electrophoresis buffer and the comb is carefully removed Prepared samples contain ing the proteins of interest are diluted with sample buffer and loaded into wells Electro phoresis is carried out for 3 to 6 hours at 4 C The gel is placed into a vertical tank transfer Co
217. hape solution viscosity and applied voltage are key factors influencing electrophoretic separation Electrophoresis is used to separate complex mixtures of proteins e g from cells subcellu lar fractions column fractions or immunopre cipitates investigate subunit compositions and verify homogeneity of protein samples Table 6 1 12 It can also serve to purify pro teins for use in further applications In poly acrylamide gel electrophoresis PAGE pro teins migrate in response to an electrical field through pores in a gel matrix consisting of polymers of cross linked acrylamide The pore size is determined by acrylamide concentra tion The combination of gel pore size and protein charge size and shape determines the migration rate of the protein Polyacrylamide gels form after polymeriza tion of monomeric acrylamide into polymeric polyacrylamide chains and cross linking of the chains by N N methylenebisacrylamide Fig 6 1 7 The polymerization reaction is initi ated by the addition of ammonium persulfate and the reaction is accelerated by TEMED which catalyzes the formation of free radicals from ammonium persulfate Because oxygen inhibits the polymerization process deaerat ing the gel solution before the polymerization catalysts are added will speed up polymeriza tion deaeration is not recommended for the gradient gel protocols because slower poly merization facilitates casting of gradient gels Precast ge
218. he remaining gels in the IEF run are extruded 7 Pour the gel and equilibration solution out of the cryovial onto a metal or plastic scoop Carefully remove excess equilibration buffer with a pipet 8 Place a few milliliters of electrophoresis buffer on the top of the second dimension gel 9 Slowly slide the IEF gel off the scoop and onto the top of the second dimension gel Remove all air bubbles trapped between the gels Remove excess electrophoresis buffer from the top of the second dimension gel The basic end of the gel may be placed on either the left or right side of the second dimen sion gel However once a convention is established all gels should be oriented the same way The acidic end of the IEF gel can be recognized in two ways the bromphenol blue will usually be yellow and a bulge increased gel diameter will be present 10 Place a piece of agarose containing molecular weight standards see Support Proto col 6 beside the basic side of the IEF gel optional Note that when molecular weight standards are used the isoelectric focusing gel has to be shorter than the width of the second dimension gel 11 Carefully overlay the IEF gel and the gel piece with standard proteins with the hot agarose equilibration buffer mixture 2 ml gel prepared in step 5 Let the agarose solidify The agarose prevents the IEF gel from shifting position and ensures good contact between the IEF and second dimension gels 12 Carefull
219. hen the protein may be at its isoelectric point or may have moved out of the gel because it had the wrong charge Con tinuous gel systems although more versatile will give lower resolution than discontinuous gels Detergents or other solubilizing agents such as urea may be needed to fully solubilize and resolve the protein Once the conditions that resolve the protein are determined Fer guson plots will give indications of multiple Current Protocols in Cell Biology subunit structure native size and potential iso form relationships Time Considerations Separations will be complete when the tracking dye or protein reaches the bottom of the gel For minigels this generally takes 1 to 2 hr using 15 or 30 mA for 0 75 or 1 5 mm thick gels respectively Standard format gels require 4 to 5 hr at 15 or 30 mA for 0 75 or 1 5 mm thick gels respectively Standard format gels can also run overnight at 4 to 6 or 8 to 12 mA for 0 75 or 1 5 mm thick gels respectively Literature Cited Andrews A T 1986 Electrophoresis Theory Tech niques and Biochemical and Clinical Applica tions 2nd ed Oxford University Press New York Ferguson K A 1964 Starch gel electrophoresis application to the classification of pituitary pro teins and polypeptides Metabolism 13 985 1002 Hames D 1990 One dimensional polyacrylamide gel electrophoresis n Gel Electrophoresis of Proteins A Practical Approach 2nd ed B D Hames and D
220. his reduces the amount of disk space necessary to store the image and the image usually will load and analyze faster with the analysis software The last step in image capture is to save the image Several options are available at this point including choosing which location to Current Protocols in Cell Biology save the image at what file type or format to use and whether to use some form of compres sion The location where the image is saved is not as trivial a question as it might seem if the image will need to be transferred to another computer at some point File sizes can easily exceed 15 megabytes on high resolution images This is a manageable size for hard drives but exceeds current floppy drive sizes by an order of mag nitude There are software utilities available that will subdivide files into disk size chunks and then reassemble them at the next computer but this is an inconvenient and slow method If the computer used to help capture the image is connected to a network the image files can easily be transferred this way or potentially saved on a central server Alternatively several types of high capacity removable media are available e g Zip or Jazz This usually re quires the installation of additional hardware onto two or more computers but does make backing up data easier Since image files can be very large com pression techniques are sometimes used to re duce disk space requirements Compression algor
221. hniques can be applied Although proteins are already visible as blue bands after BN PAGE it can be useful to stain again with Coomassie blue to reach a higher detection sensitivity Alterna tively silver staining UNIT 6 6 or immunoblot ting UNIT 6 2 are commonly used The indi vidual subunits of a complex can be identi fied by SDS PAGE or the Native Antibody Based Mobility Shift NAMOS assay see Basic Protocol 3 The NAMOS assay is a vari ant of BN PAGE in which the stoichiometry of multiprotein complexes can be determined without purification of the complex of interest Swamy et al 2007 Critical Parameters and Troubleshooting Potential problems in pouring and running acrylamide gels in general are described in UNIT 6 1 These include problems in the poly merization of the gel and critical factors when using multicasting equipment Removing the comb of the BN stacking gel might destroy the wells of the stacking gel due to the low percentage of acrylamide Try to re move the comb slowly pulling it out slightly perpendicular to the plane of the gel This al lows air to enter the wells If this does not help increase the acrylamide bisacrylamide concentration of the BN stacking gel by 0 3 If the gel pieces that form the wells are not broken but just displaced try to fix them with a syringe needle Unwanted air bubbles in the stacking gel can be aspirated using a syringe Test for leakiness with the BN cathode
222. horesis is in the determination of native protein size see Alternate Protocol Ferguson plots reviewed by Andrews 1986 were first described for starch gels Ferguson 1964 and then for polyacrylamide gels He drick and Smith 1968 Ferguson plots are prepared by separating proteins under nonde naturing conditions at several different gel con centrations As the acrylamide concentration T is increased the relative mobility R of the protein decreases This is plotted as log relative mobility on the y axis versus T on the x axis to produce a straight line The slope of this line is referred to as the retardation coefficient K and measures how effectively a protein is slowed by the increase in T Large proteins will be retarded much more signifi cantly than small proteins with increasing gel concentration with the size of the protein being proportional to the slope of the curve Once the K plots for several size standards are generated Fig 6 5 2 the K values are plotted against Current Protocols in Cell Biology the molecular weight of the standard proteins using a log log graph Fig 6 5 3 The retarda tion coefficient also depends on a large number of other variables including temperature pH buffer type ionic strength and C percent bisacrylamide cross linker All these factors should be kept constant for a given experiment In addition to estimated size other types of information are available from th
223. horesis units 9 Remove the gel tubes from the casting stand remove the Parafilm from the tube bottoms and inspect gels for irregularities or trapped air bubbles Discard imperfect gels If using gels cast with hydrostatic pressure remove the bundle of tubes en bloc cut off excess acrylamide with a razor blade and then rinse away remaining acry lamide particles from the outside of each tube Current Protocols in Cell Biology 10 Place a rubber grommet on the top of the tube Approximately 5 mm of the tube should be visible above the upper edge of the grommet 11 Mount the tube with the grommet in the upper reservoir and plug any unused holes After the tube is seated its lower end must be submerged in the lower electrode solution Be sure to remove any air bubbles trapped at the bottom of the tube by shaking or tapping the tube gently Alternatively with some units bubbles can be dislodged by raising and lowering the tubes or by using a long curved needle and syringe Prefocus the gels 12 Prepare the 0 1 M NaOH upper electrode solution by degassing under vacuum with stirring for at least 5 min The amount of upper electrode solution necessary depends on the type of electrophoresis chamber Ifa Bio Rad Protean II xi 2D apparatus is used 1 liter of 0 1 M NaOH is sufficient for both prefocusing and the separation 13 Remove the 8 M urea overlay from the top of the gels using a Pasteur pipet and place 50 ul lysis buffer on the to
224. hosphate substrate buffer 1 mM MgCl 0 1 M diethanolamine 0 02 sodium azide optional Adjust to pH 10 with HCI and use fresh Traditionally the AMPPD substrate buffer has been a solution containing 1 mM MgCl and 50 mM sodium carbonate bicarbonate pH 9 6 Gillespie and Hudspeth 1991 The use of diethanolamine results in better light output Tropix Western Light instructions Alternatively 100 mM Tris Cl pH 9 5 100 mM NaCl 5 mM MgCl can be used Sandhu et al 1991 Dioxetane phosphate visualization solution Prepare 0 1 mg ml AMPPD or CSPD Tropix or Lumigen PPD Lumigen see Table 6 2 1 substrate in dioxetane phosphate substrate buffer see recipe Prepare just before use Lumi Phos 530 Boehringer Mannheim or Lumigen is a ready to use solution and can be applied directly to the membrane This concentration of AMPPD substrate 240 uM is the minimum recommended by Tropix Western Light Ten fold lower concentrations can be used but require longer exposures Luminol visualization solution 0 5 ml 10x luminol stock 40 mg luminol Sigma in 10 ml DMSO 0 5 ml 10x p iodophenol stock optional 10 mg Aldrich in 10 ml DMSO 2 5 ml 100 mM Tris Cl pH 7 5 4PPENDIX 24 25 ul 3 H O H O to 5 ml Prepare just before use Recipe is from Schneppenheim et al 1991 Premixed luminol substrate mix Mast Immu nosystems Amersham ECL Du Pont NEN Renaissance Kirkegaard amp Perry LumiGLO may also be used p iodophenol is an optional enhanc
225. ht not be a problem Otherwise reduce the amount of detergent in the BN dialysis buffer To detect the protein of interest by Coomassie brilliant blue staining it should be present in 0 5 to 2 ug amounts If silver stain ing is used this amount can be reduced to 0 1 ug 20 to 40 ug of a protein mixture is typi cally loaded into a well of 50 ul volume on a 1 mm slab BN gel 16 cm 15 wells For im munoblotting much less protein is required depending on the quality of the antibody used for detection Current Protocols in Cell Biology Instead of giving a defined band in the first dimension or a circular spot in two dimensional BN SDS PAGE a smear that was generated in the first dimension may be seen This could have several reasons First the elec trophoresis might not have been optimal Try to improve dialysis of the sample see above If the protein migrates much higher than the dye front a shorter electrophoresis might help Second the protein might have aggregated Decrease the amount of sample loaded Do not freeze and thaw the sample Try to fur ther purify the protein of interest under na tive conditions Third the protein might be present in several overlapping complexes A two dimensional BN BN PAGE could prove whether several overlapping complexes coex ist that contain the protein of interest For de tails and other techniques to explore this pos sibility see Schamel et al 2005 Sometimes the protein is d
226. ibody based MObility Shift NAMOS assay With the NAMOS assay it is even possible to determine the stoichiometry of a protein complex that is present in low amounts within a total cell lysate Due to the large amount of added antibody detection of the protein complex of interest has to be done by immunoblotting as described in Alternate Protocols 1 and 2 and UNIT 6 2 Current Protocols in Cell Biology ALTERNATE PROTOCOL 2 BASIC PROTOCOL 3 Electrophoresis and Immunoblotting ee 6 10 11 Supplement 38 Two Dimensional Blue Native Polyacrylamide Gel Electrophoresis 6 10 12 Supplement 38 Before performing the NAMOS assay one has to set up the sample preparation Support Protocol 1 and immunodetection Alternate Protocols 1 and 2 conditions It is required that the multiprotein complex of interest be separated as a single clearly visible complex after BN PAGE Typical expected results are displayed in Figure 6 10 2 A anti X Fab anti Y Fab Figure 6 10 2 The Native Antibody Based Mobility Shift NAMOS Assay In this hypothetical experiment the protein complex of interest comprises one X and two Y subunits XY2 Its stoi chiometry is to be determined by the NAMOS assay Basic Protocol 3 Cellular lysates containing complex XY2 were separated by BN PAGE and visualized by anti XY2 immunoblotting Indeed XY2 was one defined complex lanes 1 and 7 band a In A increasing amount
227. ic bag add diluted HRPO or AP anti Ig conjugate and incubate 30 min to hr at room temperature with constant agitation When using plastic incubation trays see step 3 annotation for proper antibody solution volumes Electrophoresis 7 Remove membrane from bag and wash as in step 4 Develop according to appropriate and visualization protocol see Basic Protocol 3 or Alternate Protocol 4 Immunoblotting 6 2 9 Current Protocols in Cell Biology ALTERNATE PROTOCOL 3 Immunoblotting and Immunodetection 6 2 10 IMMUNOPROBING WITH AVIDIN BIOTIN COUPLING TO SECONDARY ANTIBODY The following procedure is based on the Vectastain ABC kit from Vector Labs see SUPPLIERS APPENDIX It uses an avidin biotin complex to attach horseradish peroxidase HRPO or alkaline phosphatase AP to the biotinylated secondary antibody Avidin bio tin systems are capable of extremely high sensitivity due to the multiple reporter enzymes bound to each secondary antibody In addition the detergent Tween 20 is a popular alternative to protein blocking agents when using nitrocellulose or PVDF filters Additional Materials also see Basic Protocol 2 Blocking buffer appropriate for membrane and detection protocol see recipe TTBS nitrocellulose or PVDF or TBS neutral or positively charged nylon see APPENDIX 24 for recipes Vectastain ABC HRPO or ABC AP AP kit Vector Labs containing the following reagent A avidin reagent B bio
228. ich stock solution 500 mM final 40 ml 1 M NaCl 20 mM final 4 ml 0 5 M EDTA pH 8 0 APPENDIX 24 2 mM final 200 ml glycerol 10 v v final H20 to 1 liter Store at room temperature stable at least 1 year Preparation of an EDTA stock solution is described in UNIT 6 4 BN stacking gel solution 3 ml 3x BN gel buffer see recipe 0 72 ml acrylamide bisacrylamide mix see recipe 5 28 ml H2O 120 ul 10 w v aqueous ammonium persulfate add immediately before pouring gel 12 ul TEMED add immediately before pouring gel This will result in a 3 2 solution Adjust volumes as necessary Denaturing BN transfer buffer 5 81 g Tris base 48 mM final 2 93 g glycine 39 mM final 200 ml methanol 20 final 1 g SDS 0 1 final H20 to 1 liter Store at 4 C stable at least 1 year Destaining solution 450 ml methanol 45 v v final 100 ml acetic acid 10 v v final H20 to 1 liter Store at room temperature stable at least 1 year Detergent stock solutions 2x digitonin Prepare a 2 w v solution of digitonin Sigma Aldrich in water by heating to 95 C Store in 1 to 10 ml aliquots up to 5 years possibly longer at 20 C Thawed solutions are stable at room temperature for up to 1 week If a precipitate forms reheat the solution to 95 C until the digitonin redissolves 10x Brij 96 Prepare a 10 w v solution of Brij 96 e g Brij 96V in water Store up to 1 year possibly longer at room temperatur
229. id potential reproducibility problems all samples should be processed identically Prepare the samples for isoelectric focusing 18 19 20 If cell extracts were frozen thaw samples and immediately add dry urea to 9 M final concentration The amount of urea in mg equals 0 83 times the sample volume in ul For example use 83 mg urea per 100 ul sample The final volume of the sample with urea added equals 1 6 times the initial volume 160 ul in the same example Add an equal volume of lysis buffer 160 ul in the above example and warm briefly if necessary to dissolve urea Filter samples using a 0 2 um microcentrifuge filter unit by microcentrifuging at maximum speed room temperature until the entire sample has passed through the filter Load the desired volume onto the IEF gel Ifa 500 ug total protein load per 3 mm gel is desired a practical maximum load for most whole cell extracts the protein concentration determined during the protein assay has to be 25 ug ul If the sample is less concentrated the sample volume required will be too large for a3 mm IEF gel Alternatively sample loads can be based on cell numbers radioactivity or any other appropriate reference see Basic Protocol 1 step 22 Current Protocols in Cell Biology Electrophoresis and Immunoblotting 6 4 19 Supplement 4 SUPPORT PROTOCOL 5 BASIC PROTOCOL 3 Two Dimensional Gel Electrophoresis 6 4 20 Supplement 4
230. ides Among these dyes which included amido black 10B and fast green FCF the Coomassie blue dyes gave higher sensitiv ity Wilson 1979 and became routinely used A large number of staining protocols using Coomassie brilliant blue R 250 as a dye have been published see Neuhoff et al 1985 and references therein The protocol described in this unit Basic Protocol 1 involves simulta neous protein fixation and staining in a solu tion containing the dye in methanol acetic acid water followed by removal of the un bound dye by washing with methanol acetic acid water This is the most commonly used protocol for protein staining in gels Proposed variations to this procedure include using no methanol to retain small polypeptides Schag ger and von Jagow 1987 decreasing the con centrations of both methanol and acetic acid to limit protein fixation Rosenfeld et al 1992 and using ion pairing agents to reduce back ground Choi et al 1996 Protocols using a colloidal form of Coomassie brilliant blue G 250 which rival in sensitivity those using Coomassie brilliant blue R 250 have also been described see for example Neuhoff et al 1985 DeSilva 1995 A dramatic increase in sensitivity was achieved with the introduction of protein stain ing methods based on the selective reduction of silver ions to form metallic silver images Switzer et al 1979 The method relies on the autocatalytic reduction of silver a key phe no
231. idly degrade the sample thus first heating the samples to 70 to 100 C for 3 min is recommended In some cases heating to 100 C in sample buffer will cause selective aggregation of pro teins creating a smeared layer of Coomassie blue stained material at the top of the gel Gallagher and Leonard 1987 To avoid heat ing artifacts and also prevent proteolysis the use of specific protease inhibitors during protein isolation and or lower heating tem peratures 70 to 80 C have been effective Dhugga et al 1988 Although continuous gels suffer from poor band sharpness they are less prone to arti facts caused by aggregation and protein cross linking If streaking or aggregation appear to be a problem with the Laemmli system then the same sample should be subjected to con tinuous SDS PAGE to see if the problem is intrinsic to the Laemmli gel or the sample If the protein bands spread laterally from gel lanes the time between applying the sam ple and running the gel should be reduced in order to decrease the diffusion of sample out of the wells Alternatively the acrylamide per centage should be increased in the stacking gels from 4 to 4 5 or 5 acrylamide or the operating current should be increased by 25 to decrease diffusion in the stacking gel Use caution when adding 1x SDS electrophoresis buffer to the upper buffer chamber Samples Current Protocols in Cell Biology can get swept into adjacent wells and onto
232. iline DryStrip kit Amersham Pharmacia Biotech including Cathode electrode Anode electrode Sample cup bar Tray Sample cups Immobiline strip aligner IEF electrode strips Sample application pieces Instruction manual continued Current Protocols in Cell Biology Protein sample to be analyzed Lysis buffer see recipe Immobiline DryStrip reswelling tray Amersham Pharmacia Biotech Forceps Filter paper Glass plate Flatbed electrophoresis unit Amersham Pharmacia Biotech Multiphor II or equivalent Recirculating cooling water bath Power supply minimum capacity of 3000 to 3500 V Petri dishes Additional reagents and equipment for protein detection by staining APPENDIX 3 and or for electroblotting UNIT 6 2 optional Rehydrate the Immobiline DryStrip s 1 Prepare an appropriate rehydration solution for the type of DryStrips to be used as described in Table 6 4 1 400 ul rehydration solution per 18 cm DryStrip The rehydration solution should contain 7 Murea 2 M thiourea and 2 CHAPS or another appropriate detergent zwitterionic or nonionic such as Triton X 100 NP 40 or n octyl glucoside should be included in the rehydration solution to aid in sample solubility The optimal detergent and detergent concentration may vary with type of sample and should be determined empirically One possible method of loading large sample volumes onto IPG gels is to add the sample directly to the rehydration solution Sample loading dur
233. imal for immunization Also the use of native gels allows separation and recovery of nondenatured proteins for func tional studies The electrophoresis procedure outlined above illustrates the ability of agarose to sepa rate proteins of molecular weights that exceed Current Protocols in Cell Biology e 900 kDa gt 700 kDa gt 1 50 204 kDa gt as 1 36 4 1 26 7 1 135 1 015 0 89 5 0 77 5 0 64 0 52 5 Optical density 0 40 T T T o 10 20 30 40 Millimeters 50 60 70 80 90 100 Figure 6 7 2 Luminograph of vWF multimers from normal plasma lane 1 von Willebrand disease Type 2B plasma lane 2 and von Willebrand disease Type 2A plasma lane 3 A densitometric tracing is seen below Reproduced with permission from Krizek and Rick 2000 1 x 10 Da For smaller proteins higher con centrations of agarose e g 3 and shorter blotting times may be used In instances where electroblotting cannot be carried out because of precipitation of the proteins during transfer due to separation from detergent the proteins can be immobilized in the gel to prevent diffu sion before immuno identification Immobi lization of the proteins also allows for the use of sequential antibodies for identification of protein bands Additionally in gel identifica tion may also be important if there is uneven transfer of proteins due to dissimilar transfer characteristics Immobilization of th
234. ime Times vary from 1 to 5 days It is helpful to secure the gel to the cassette with tape to keep it from changing position Current Protocols in Cell Biology Electrophoresis and Immunoblotting 6 7 7 Supplement 15 Agarose Gel Electrophoresis of Proteins 6 7 8 Supplement 15 REAGENTS AND SOLUTIONS Use deionized or distilled water in all recipes and protocol steps For common solutions see APPENDIX 2A for suppliers see SUPPLIERS APPENDIX Agarose gel buffer 0 05 M Na HPO 0 1 w v SDS Adjust pH to 7 0 with concentrated HC1 Filter and store up to 3 months at room temperature Agarose running buffer 0 1 M Na HP0 0 1 w v SDS Adjust pH to 7 0 with concentrated HCl Store up to 3 months at room temperature Blocking buffer in gel 15 ml 16 6 M ethanolamine 1 0 M 250 mg fatty acid free Fraction V BSA Add H O to 200 ml Adjust pH to 8 0 with concentrated HCl Add H O to 250 ml Prepare fresh on the day of use Borate saline buffer BSB 15 4 g boric acid 36 mM 65 06 g NaCl 143 mM 1 40 g NaOH 0 005 N Add 1 56 ml concentrated HCI to adjust pH to 7 83 Adjust volume to 7 liters with H O Store up to 3 months at room temperature Electrophoresis buffer 1x Dilute 10x TAE see recipe 1 10 in water Add 10 ml of 20 w v SDS APPENDIX 2A per 2 liters Final concentrations are 40 mM Tris acetate 1 mM EDTA and 0 1 w v SDS Final pH is 7 8 to 8 3 Store up to 1 week at room temperature
235. imensional analysis the first di mension separation is performed in a single column or lane and then a second separation is performed perpendicular to the first The result after visualization is a rectangular image of up to 10 000 spots The most common two dimen sional gel type is one in which protein is sepa rated first by apparent pI and second by mo lecular weight although two dimensional separation of nucleic acids is also possible While many of the concepts and analysis tech niques used with one dimensional gels are ap plicable to two dimensional gels the complex nature of most two dimensional gels requires somewhat different methodology For example spots are more difficult to detect since they are not conveniently arranged in lanes and can vary more in shape and overlap than bands In addi tion two dimensional experiments usually re quire some method of comparing between two images whereas one dimensional images usu ally contain all of the information from an experiment Spot detection Probably the most difficult aspect of two di mensional analysis is efficient and accurate spot detection If it is incorrectly done it can lead to hours of manual editing Due to the complexity and computational intensity of some algorithms the detection process itself can last hours on relatively fast desktop com puters One theoretically effective but compu tationally intense method is to treat the image as essentially a thre
236. in a plastic bag and stored for up to I week Load and run the gel 15 Prepare the protein sample and protein molecular weight standards mixture Load and run the gel see Basic Protocol 1 steps 13 to 26 The gel can be stained with Coomassie blue or silver UNIT 6 6 16 After staining dry the gels onto Whatman 3MM or equivalent filter paper Gradient gels gt 0 75 mm thick require special handling during drying to prevent cracking The simplest approach to drying gradient gels is to use thin gels lt 0 75 mm gradient gels with lt 20 acrylamide solutions will dry without cracking as long as the vacuum pump is working properly and the cold trap is dry at the onset of drying For gradient gels gt 0 75 mm thick add 3 w v glycerol to the final destaining solution to help prevent cracking Another method is to dehydrate and shrink the gel in 30 methanol for up to 3 hr prior to drying Then place the gel in distilled water for 5 min before drying CASTING MULTIPLE GRADIENT GELS Casting gradient gels in a multiple gel caster has several advantages In addition to the time savings batch casting produces gels that are essentially identical This is particu larly important for gradient gels where slight variations in casting technique can cause variations in protein mobility The gels may be stored for up to 1 week after casting to ensure internal consistency from run to run during the week Furthermore gels with several ranges of co
237. in at room temperature A sharp optical discontinuity at the overlay gel interface will be visible on polymeriza tion Failure to form a firm gel usually indicates a problem with the ammonium persulfate TEMED or both Ammonium persulfate solution should be made fresh before use Am Electrophoresis monium persulfate should crackle when added to the water If not fresh ammonium and i persulfate should be purchased Purchase TEMED in small bottles so if necessary a new Immunoblotting 6 1 5 previously unopened source can be tried Current Protocols in Cell Biology Supplement 37 One Dimensional SDS PAGE 6 1 6 Supplement 37 Table 6 1 1 Recipes for Polyacrylamide Separating and Stacking Gels SEPARATING GEL Final acrylamide concentration in separating gel Stock solution 5 6 7 7 5 8 9 10 12 13 15 30 w v acrylamide 2 50 3 00 3 50 3 75 4 00 4 50 5 00 6 00 6 50 7 50 0 8 w v bisacrylamide 4x Tris Cl SDS ZID 2319 35 3S 3I SAD 3 31D 313 BIS pH 8 8 H20 8 75 8 25 7 75 7 50 7 25 6 75 6 25 5 25 4 75 3 75 10 w v ammonium 0 05 0 05 0 05 0 05 0 05 0 05 0 05 0 05 0 05 0 05 persulfate TEMED 0 01 0 01 0 01 0 01 0 01 0 01 0 01 0 01 0 01 0 01 Preparation of separating gel In a 25 ml side arm flask mix 30 acrylamide 0 8 bisacrylamide solution 4x Tris Cl SDS pH 8 8 see reagents below and H20 Degas under vacuum 5 min Add 10 ammonium persulfate and TEMED Swirl gen
238. ing agent that increases light output Luminol and p iodophenol stocks can be stored for lt 6 months at 20 C Ponceau S solution Dissolve 0 5 g Ponceau S in 1 ml glacial acetic acid Bring to 100 ml with water Prepare just before use Electrophoresis and Immunoblotting 6 2 15 Current Protocols in Cell Biology Immunoblotting and Immunodetection 6 2 16 Transfer buffer Add 18 2 g Tris base and 86 5 g glycine to 4 liters of water Add 1200 ml methanol and bring to 6 liters with water The pH of the solution is 8 3 to 8 4 For use with PVDF filters decrease methanol concentration to 15 for nylon filters omit methanol altogether CAPS transfer buffer can also be used Add 2 21 g cyclohexylaminopropane sulfonic acid CAPS free acid 0 5 g DTT 150 ml methanol and water to 1 liter Adjust to pH 10 5 with NaOH and chill to 4 C For proteins gt 60 kDa reduce methanol content to 1 Moos 1992 COMMENTARY Background Information Immunoprecipitation has been widely used to visualize the antigens recognized by various antibodies both polyclonal and monoclonal unit 7 2 However there are several problems inherent with this method including the re quirement for radiolabeling of antigen co pre cipitation of tightly associated macromole cules occasional difficulty in obtaining pre cipitating antibodies and insolubility of various antigens Talbot et al 1984 To circumvent these problems elec
239. ing rehydration is preferred when using the IPGphor system with its single strip holder for both rehydration and isoelectric focusing Solutions containing urea should be filtered using a 0 2 um filter before use 2 Slide the protective lid off the reswelling tray and level the tray by adjusting the leveling feet until the leveling bubble is centered 3 For an 18 cm gel pipet 350 to 400 ul of rehydration solution into a slot of the reswelling tray Move the pipet along the length of the well while adding the solution to spread it evenly throughout the length of the slot Avoid excessive air bubble formation while pipetting this solution If the IPGphor system is used pipet the rehydration solution containing the protein sample into each cleaned ceramic holder 4 Remove the protective cover from the Immobiline DryStrips and gently place them gel side down into the prepared slot To facilitate their removal after rehydration the strips should be oriented with their pointed ends at the sloped end of the slots in the rehydration tray Be careful not to trap any air bubbles under the gel strips 5 Overlay each strip with 2 to 3 ml of DryStrip cover fluid to prevent evaporation and urea crystallization Slide the protective lid into place and allow gels to rehydrate overnight 16 hours at room temperature Shorter rehydration times can be used although a minimum of 6 to 8 hr is usually needed to completely and reproducibly rehydrate
240. ing the assembly of the head of bacteriophage T4 Nature 227 680 685 O Farrell P H 1975 High resolution two dimensional polyacrylamide gel electrophoresis of proteins J Biol Chem 250 4007 4021 Juan S Bonifacino Current Protocols in Cell Biology Electrophoresis and Immunoblotting 6 0 3 Supplement 15 One Dimensional SDS Gel Electrophoresis of Proteins Sean R Gallagher UVP Inc Upland California ABSTRACT Electrophoresis is used to separate complex mixtures of proteins e g from cells subcel lular fractions column fractions or immunoprecipitates to investigate subunit compo sitions and to verify homogeneity of protein samples It can also serve to purify proteins for use in further applications In polyacrylamide gel electrophoresis proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix pore size decreases with increasing acrylamide concentration The combination of pore size and protein charge size and shape determines the migration rate of the protein In this unit the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions that is in the presence of sodium dodecyl sulfate SDS Both full size and minigel formats are detailed Several modifications are provided for specific applications For separation of peptides and small proteins the standard buffers are re placed with either a Tris tricine buffer s
241. ins and therefore might be used as standards Schagger et al 1994 Second protein glycosylation and phospho rylation might alter the running behavior of proteins in BN PAGE Note that the marker proteins are usually non transmembrane non glycosylated and non phosphorylated pro teins Especially if you work with trans membrane proteins you cannot deduce the molecular weight of the proteins from BN PAGE Third in addition to their molecu lar weight proteins are also separated ac cording to their shape and number of bound Coomassie blue molecules Thus proteins that deviate significantly from a ball like shape and very basic proteins show reduced mobility Lastly it might be that the expected molec ular weight value of the protein complex is wrong Many antibodies that work well for the immunodetection western blotting of your protein of interest after SDS PAGE do not recognize the protein after the first dimension BN PAGE In the author s experience the re moval of Coomassie blue from the blotting membrane does not help Try both condi tions for the transfer to the blotting membrane Alternate Protocols 1 and 2 If this does Electrophoresis and Immunoblotting 6 10 19 Supplement 38 Two Dimensional Blue Native Polyacrylamide Gel Electrophoresis 6 10 20 Supplement 38 not help then a two dimensional BN SDS PAGE must be performed In any case one should verify immunoblotting results
242. ins precipitate with those ions and consequently do not enter the gel Hence cations that interact with Coomassie blue have to be removed and substituted by 6 aminohexanoic acid in order to maintain a certain ionic strength of the solution which is necessary for the solubility and stabilization of many protein complexes Sodium ions are tolerated to a maximum concentration of 50 mM Third samples have to be loaded with detergent on BN gels Otherwise proteins aggregate during the stacking step of the electrophoresis Membrane and organelle fractions lysed in BN lysis buffer can be directly applied to BN gels Cellular lysates have to be dialysed against BN dialysis buffer in order to remove small cations and metabolites Proteins bound to a matrix are washed and eluted in BN dialysis buffer This protocol describes the preparation of cell lysates Cells are washed and lysed in any lysis buffer although we recommend using the BN lysis buffer After removal of insoluble material the lysate is dialysed against BN dialysis buffer In this protocol a self made dialysis setup utilizing dialysis membranes is described Alternative desalting methods can be applied as well Materials Cell culture dish 10 to 15 cm containing cells of interest 80 confluent Phosphate buffered saline PBS see recipe ice cold BN lysis buffer see recipe BN dialysis buffer see recipe Cell scrapers 10 to 50 ml centrifuge tubes Refrigerated cell cultur
243. ion method electronic noise and other factors Since this background tends to be nonuniformly distributed through out the image failure to subtract it can make band detection and quantitation less accurate Many methods of background subtraction are possible Sometimes it is possible to generate a second image under conditions that do not detect the protein or nucleic acid The second image is then digitally subtracted from the data containing image to remove the back ground More often background information must be obtained from a single image If the background varies uniformly across the image a line that crosses the variation can be defined at a point where no bands are present The intensity values at each point on the line can be used as the background value for the pixels perpendicular to the line at that point Com monly background is also present as variations in intensity along the long axis within each lane One simple method is to take the lowest point in the lane profile as the background Another Current Protocols in Cell Biology method is to use an average value of the edge of each band as the background for that band More complicated methods such as valley to valley and rolling disk use local minima points in the lane profile to define a variable back ground along the length of the lane Because there can be many different causes and distri butions of background no single method of background determination can
244. ipet tip might be too wide to fit between the glass plates 8 Prepare water saturated isobutyl alcohol by shaking isobutyl alcohol and water in a glass bottle Using a Pasteur pipet overlay the separating gel with water saturated pale a isobutyl alcohol upper alcoholic phase from the mix by gently layering the alcohol Immunoblotting 6 10 3 Current Protocols in Cell Biology Supplement 38 Table 6 10 1 Gel Solutions for BN PAGE Low BN Stacking separating High BN separating gel solution gel gel solution solution 4 1 10 12 16 18 3 2 40 acrylamide 3 00 5 25 7 50 9 00 12 00 13 50 1 00 bisacrylamide mix ml 3x BN gel buffer ml 10 00 10 00 10 00 10 00 10 00 10 00 4 17 dH20 ml 17 00 7 33 70 v v glycerol ml 14 75 12 50 11 00 8 00 6 50 10 ammonium 108 84 84 84 84 84 167 persulfate ul TEMED ul 11 8 8 8 8 8 17 aNumbers in the body of the table are milliliters of stock solution except 10 APS and TEMED which are microliters and should be added only when the low and high percentage BN gel solutions are already in the chambers of the mixing apparatus gt See recipe in Reagents and Solutions Two Dimensional Blue Native Polyacrylamide Gel Electrophoresis 6 10 4 Supplement 38 against the edge of one and then the other of the spacers to produce a smooth surface In the multicasting equipment all gels must be overlaid with the same volume of water saturated isobutyl alcohol
245. ir staining results in the same band intensity per protein mass unit This assumption is not necessarily correct es pecially for Coomassie blue or silver staining where band intensities are known to be influ enced by the proteins amino acid composi tions Another limitation is that measurements should be made only within a linear range Staining with SYPRO dyes which interact with the protein SDS complex rather than protein functional groups has been reported to give relatively less protein to protein variability and Molecular mass kDa Molecular mass kDa 14 l l l l l i l l l ng per 1000 300 100 30 10 3 1 1000 300 100 30 10 3 1 band Figure 6 6 1 Comparison of the protein staining methods described in this unit Serial dilutions of a mixture of rabbit muscle myosin 205 kDa rabbit muscle phosphorylase b 97 kDa bovine serum albumin 66 kDa chicken ovalbumin 45 kDa bovine erythrocyte carbonic anhydrase 29 kDa and chicken egg lysozyme 14 kDa were resolved by SDS PAGE on a 4 to 20 T gradient gel and stained with A Coomassie blue Basic Protocol 1 B silver Basic Protocol 2 C SYPRO Ruby Basic Protocol 3 or D zinc Basic Protocol 4 The amounts in nanograms per band loaded on each lane are indicated at the bottom of the figure Images of stained gels were acquired on a Bio Rad Gel Doc 1000 gel documentation system using a standard UV 300 nm transilluminator
246. irculating water bath are placed into and the transfer unit for cooling Immunoblotting 6 2 3 Current Protocols in Cell Biology 12 Turn off the power supply and disassemble the apparatus Remove membrane from blotting apparatus and note orientation by cutting a corner or marking with a soft lead pencil or Paper Mate ballpoint pen Many ballpoint inks come off but Paper Mate stays on the membrane Membranes can be dried and stored in resealable plastic bags at 4 C for 1 year or longer at this point Prior to further processing dried PVDF membranes must be placed into a small amount of 100 methanol to wet the membrane then in distilled water to remove the methanol 13 Stain gel for total protein with Coomassie blue to verify transfer efficiency If desired stain membrane reversibly to visualize transferred proteins see Support Protocol 1 or irreversibly with Coomassie blue India ink naphthol blue or colloidal gold These staining procedures are incompatible with nylon membranes If membrane shows significant staining on the backside either the gel was heavily overloaded or the membrane has poor protein binding capacity see Troubleshooting In either case protein binding sites on the side facing the gel are saturated allowing protein to migrate to the other side of the membrane Nitrocellulose in particular will show diminished binding capacity with age or poor storage conditions e g high temperature and humidity In a
247. is poured before polymerization occurs See UNIT 6 1 for instruc tions for using gradient makers 5 Do not disturb the gel during the first 10 min to allow the gradient to stabilize Allow the gel to polymerize 1 hr in a 50 C oven Dry and store the gel 6 Allow the gel to cool to room temperature then disassemble the cassette Cut off a small corner to label the anode end of the gel 7 Wash the gel 2 to 3 hr with 200 to 300 ml water Use an orbital shaker and change the water two or three times At this stage the gel may be used immediately if no additives are needed or dried as described in steps 8 to 10 8 Wash the gel 30 min to 1 hr with 200 to 300 ml of 2 5 v v glycerol 9 Place the gel on a glass plate gel side up in a dust free environment and allow to dry at room temperature overnight 10 Store the dried gel in a sealed plastic bag at 20 C for up to 2 months The gels may be rehydrated when needed see Support Protocol 2 PREPARING TISSUE CULTURE CELL EXTRACTS FOR ISOELECTRIC FOCUSING Preparation of samples containing relatively pure proteins for isoelectric focusing is generally straightforward see Basic Protocol 1 step 22 In contrast complex samples such as whole cell extracts tissue extracts or subcellular fractions are more difficult to prepare for successful isoelectric focusing Solubility limitations both prior to isoelectro focusing and during focusing restrict analysis of these complex sample
248. ish peroxidase HRPO or alkaline phosphatase AP anti Ig conjugate Cappel Vector Labs Kirkegaard amp Perry or Sigma dilute as indicated by manufacturer and store frozen in 25 1 aliquots until use Heat sealable plastic bag Powder free gloves Plastic box 1 Place membrane in heat sealable plastic bag with 5 ml blocking buffer and seal bag Incubate 30 min to 1 hr at room temperature with agitation on an orbital shaker or rocking platform Usually 5 ml buffer is sufficient for two to three membranes 14 x 14 cm size If membrane is to be stripped and reprobed see Support Protocol 3 blocking buffer must contain casein for AP systems or nonfat dry milk Plastic incubation trays are often used in place of heat sealable bags and can be especially useful when processing large numbers of strips in different primary antibody solutions 2 Dilute primary antibody in blocking buffer Primary antibody dilution is determined empirically but is typically 1 100 to 1 1000 for a polyclonal antibody Fig 6 2 3 1 10 to 1 100 for hybridoma supernatants and 21 1000 Current Protocols in Cell Biology Serum dilution S66 Ss Figure 6 2 3 Serial dilution of primary ess 8 Ss SS antibody directed against the 97 kDa or A Fy O re O A a e a catalytic subunit of the plant plasma M kDa membrane ATPase Blot was developed with 200 HRPO coupled avidin biotin reagents 116 according to the second alternate protocol 97 an
249. isualize the protein bands by placing the polyacrylamide gel on a dark black or blue surface The stained gel can be kept in water for several weeks at 4 C Solubilize the SDS zinc precipitate optional 9 Incubate the gel with 10 gel vol E Zinc Eraser with gentle agitation until the gel matrix is completely clear 5 to 10 min These two steps involve solubilization of the SDS zinc precipitate to completely destain the gel thus allowing further analysis of the protein band s of interest 10 Rinse the gel with distilled water At this point the gel is ready for downstream applications e g electroelution im munoblotting etc REAGENTS AND SOLUTIONS Use HPLC grade or Milli Q purified water or equivalent in all recipes and protocol steps For common stock solutions see APPENDIX 24 for suppliers see SUPPLIERS APPENDIX Coomassie blue staining solution For 100 ml Dissolve 0 1 g Coomassie brilliant blue R 250 in a mixture of 30 ml methanol and 20 ml water Subsequently add 40 ml of water and 10 ml of glacial acetic acid Store for up to 4 months at room temperature CAUTION Glacial acetic acid and methanol are volatile and toxic and should be handled in a chemical fume hood This solution is 0 1 w v Coomassie brilliant blue R 250 in methanol acetic acid water 3 1 6 vA Developer solution Dissolve 6 g Na CO in 98 ml water Add 50 ul of 37 formaldehyde and 2 ml of thiosulfate solution see recipe Prepare
250. ith standard protein s Downstream applications Although PAGE was initially used only for analytical purposes a large variety of micro techniques have been developed that use PAGE as a preparative step Proteins separated by one or two dimensional PAGE can be treated in gel with proteases Rosenfeld et al 1992 or cy anogen bromide Cordoba et al 1997 to ob tain peptide fragments for microsequencing electroeluted or transferred to nitrocellulose or poly vinylidene difluoride PVDF mem branes unr 6 2 for further analysis such as immunodetection or Edman degradation Pro teins separated by nondenaturing PAGE unr 6 5 or sometimes even by SDS PAGE can be analyzed in gel for enzymatic or ligand bind ing activity Both the SYPRO Ruby and nega tive zinc staining methods are compatible with virtually all of these applications A notable exception is activity analysis of proteins sepa rated by nondenaturing PAGE which are sensi tive to SDS as both staining procedures require the presence of SDS for band visualization On the other hand Coomassie blue and silver gel staining involve protein fixation and therefore are compatible with fewer downstream appli cations It is worth mentioning however that Coomassie stained proteins can be efficiently electrotransferred to PVDF membranes for Ed man degradation sequencing U Hellman pers commun although for immunoblot analysis the Coomassie blue dye is a known interf
251. ith nonlinear gradient gels Care must be taken with multiple curve techniques since they rely on only a few data points for any one part of the composite curve and outlying data points can drastically affect the outcome For size and R determination a uniform position must be found in each lane as a point from which to measure the mobility of each band Many software analysis packages use the end of the lane as the measuring start point so for them it is important to position each lane start point at an iso molecular weight or iso R point A convenient point is the well or sample loading position since it is usually easily de Electrophoresis and Immunoblotting 6 9 9 Supplement 16 Digital Electrophoresis Analysis 6 9 10 Supplement 16 tectable and at an equal mobility position in each lane A consistent point on each band must also be chosen to measure mobility A band s peak is easily defined in digital image analysis and is commonly used Since peak positions are harder to detect visually than edges on silver halide images the leading band edge is some times used when comparing digital results with silver halide based results Once lanes and bands have been detected it is also possible to quantify the amount or at least relative amount of nucleic acid or protein present in each band The amount in a band is related to the sum total of the intensity values of each pixel subtracted by the backg
252. ithms use several methods typically by replacing frequent or repetitive values or pat terns with smaller reference values and by re placing pixel values with the smaller difference values describing the change in adjacent pixels When the file is later decompressed the com pressed values are then replaced with the origi nal information Not all images compress equally with simple images containing mostly repetitive motifs compressible by 290 while complex images will benefit much less from compression Because compression is a much slower method of saving files and will not benefit every file compression is not used to save all files Several different forms of com pression are available but are separable into two main classes lossless and lossy Lossless meth ods faithfully and completely restore the image when it is decompressed no loss of data but offer only moderate file compression compres sion values range from 10 to 90 depend ing on the image Examples of lossless com pression include Huffman coding Huffman 1952 RLE Run Length Encoding and LZW Lempel Ziv and Welch Welch 1984 In comparison lossy methods such as JPEG Joint Photographic Experts Group MPEG Moving Picture Experts Group or fractal compression schemes can reach compression values of 298 Russ 1995 The trade off is that not all Current Protocols in Cell Biology information from the original file is recovered during decompressio
253. ive electrophoresis is to leave out the SDS and reducing agent DTT from the standard Laemmli SDS PAGE protocol UNIT 6 1 The gels are prepared as described in uniT 6 5 except that the sample buffer contains no SDS or DTT samples are not heated and the gel and electrophoresis solutions are prepared without SDS This protocol illustrates the separation of standard proteins at four different concentrations of acrylamide and how the results are used to construct a molecular weight standard curve Ferguson plot without the need for SDS By plotting relative mobility against T percentage weight per volume of acrylamide plus bisacry lamide in the gel the presence of isoforms and multimeric proteins can also be detected Materials 4x Tris Cl pH 8 8 1 5 M Tris Cl APPENDIX 24 4x Tris Cl pH 6 8 0 5 M Tris Cl Protein sample of interest 2x Tris glycerol sample buffer see recipe Current Protocols in Cell Biology ALTERNATE PROTOCOL Electrophoresis and Immunoblotting 6 5 5 Supplement 5 One Dimensional Electrophoresis Using Nondenaturing Conditions 6 5 6 Supplement 5 Native protein standards e g Sigma nondenatured protein molecular weight kit Tris glycine electrophoresis buffer see recipe Assemble the glass plate sandwich of the gel electrophoresis unit and place it in the casting stand Prepare and cast the gels using 4x Tris Cl pH 8 8 for the separating gel and 4x Tris Cl pH 6 8 fo
254. ively lay out a square of plastic wrap and pipet I to 2 ml visualization solution into the middle Place membrane on the plastic so that the visualization solution spreads out evenly from edge to edge Fold wrap back onto membrane seal and proceed to step 5 Remove membrane drain and place face down on a sheet of clear plastic wrap Fold wrap back onto membrane to form a liquid tight enclosure To ensure an optimal image only one layer of plastic should be between the membrane and film Sealable bags are an effective alternative Moisture must not come in contact with the X ray film Ina darkroom place membrane face down onto film Do this quickly and do not reposition a double image will be formed if the membrane is moved while in contact with the film A blurred image is usually caused by poor contact between membrane and film use a film cassette that ensures a tight fit Expose film for a few seconds to several hours Typically immunoblots produce very strong signals within a few seconds or minutes However weak signals may require several hours to an overnight exposure If no image is detected expose film 30 min to I hr and if needed overnight see Troubleshooting If desired wash membrane in two 15 min washes of 50 ml TBS and process for chromogenic development see Basic Protocol 3 Chemiluminescent and chromogenic immunoblotting can be easily combined on a single blot to provide a permanent visual marker of a
255. ize proteins using any method available The most commonly used methods are transfer to a membrane followed by immunode tection western blotting UNIT 6 2 or staining with silver or Coomassie brilliant blue UNIT 6 6 Optionally stained spots can be cut out and the protein identified by mass spectroscopy DENATURING TRANSFER OF THE PROTEINS FROM THE FIRST DIMENSION BN PAGE GEL TO A MEMBRANE FOR IMMUNOBLOTTING Protein complexes separated by BN gels can be visualized by immunoblotting also referred to as western blotting without a second dimension separation If a cell lysate is analyzed a very specific antibody is necessary that does not cross react with other proteins It is recommended to first test the quality of the antibody by second dimension BN SDS PAGE see Commentary In general the same antibodies that are used for immunodetection after SDS PAGE are potentially suitable In this protocol the proteins multiprotein complexes are first denatured within the BN gel by boiling in SDS Subsequent steps including the transfer to a membrane and the immunoblotting procedure are similar to the standard protocol used after SDS PAGE UNIT 6 2 If PVDF membranes are used the bound Coomassie blue can partially be removed This is not the case for nitrocellulose membranes that can be used to immobilize the proteins after BN PAGE as well Materials First dimension BN PAGE gel Basic Protocol 1 Phosphate buffered saline PBS see reci
256. k of the GelBond under a stream of water before proceeding to protein detection Either Coomassie blue or silver staining may be used but silver staining produces particularly fine resolution with thin GelBond backed gels Compared to staining thicker gt 0 75 mm gels thin lt 0 75 mm gels stain and destain more quickly Although the optimum staining times must be empirically determined all steps in Coomassie blue and silver staining procedures are generally reduced by half CASTING MULTIPLE SINGLE CONCENTRATION GELS Casting multiple gels at one time has several advantages All the gels are identical so sample separation is not affected by gel to gel variation Furthermore casting ten gels is only slightly more difficult than casting two gels Once cast gels can be stored for several days in a refrigerator Additional Materials also see Basic Protocol 1 Separating and stacking gels for single concentration gels Table 6 1 9 Multiple gel caster Bio Rad Hoefer 100 ml disposable syringe and flat tipped needle Extra plates and spacers 14 x 14cm acrylic blocks or polycarbonate sheets 250 and 500 ml side arm flasks used in gel preparation Long razor blade or plastic wedge Wonder Wedge Hoefer Resealable plastic bags Pour the separating gel 1 Assemble the multiple gel caster according to the manufacturer s instructions With the Hoefer unit make sure to insert the large triangular space filler plug in the base of th
257. known protein First probe membrane with the chemiluminescent reactions to record on film If stripping and reprobing is needed then process by wetting and NaOH treatment see Support Protocol 3 For the last reaction use chromogenic development to produce a permanent visual record of the blot Alterna tively once the film record of the chemiluminescent blot is recorded the blot can be rinsed briefly with distilled water and placed in the appropriate chromogenic solution for chromogenic development of the blot This results in a permanent reference stain on the blot for comparison to the more easily scanned and quantitated film record Current Protocols in Cell Biology Electrophoresis and Immunoblotting 6 2 13 SUPPORT PROTOCOL 3 Immunoblotting and Immunodetection ee 6 2 14 STRIPPING AND REUSING MEMBRANES This stripping procedure works with blotted membranes from one and two dimensional gels as well as with proteins blotted from previously stained gels Suck and Krupinska 1996 Reprobing PVDF membranes that have been developed with chemiluminescent reagents is simple and straightforward All residual antibodies are removed from the membrane by first rewetting it in water and then briefly treating it with NaOH Although repeated reprobing can lead to loss of signal up to five reprobings generally are feasible The blot should have been previously blocked with 5 nonfat dry milk prior to treatment Materials 0 2 M
258. l 0 2 EDTA 0 15 mM PMSF lug ml leupeptin and 1 ug ml pepstatin Pepstatin I mg ml 10 mg pepstatin 10 ml anhydrous ethanol Divide into convenient volumes Store at 20 C stable at least 1 year Thaw aliquots and mix well immediately before use Reducing buffer 0 5 g dithiothreitol DTT 0 1 g SDS 1 51 g Tris base Adjust to pH 6 8 with HCl Add H O to 100 ml Prepare fresh every time Final concentrations are 0 5 w v DTT 0 1 w v SDS and 125 mM Tris Cl pH 6 8 Tris SDS buffer 0 3 g SDS 0 6 g Tris base Adjust to pH 8 0 with HCl Add H O to 100 ml Divide into 5 ml aliquots Store at 80 C stable at least 1 year Final concentrations are 0 3 w v SDS and 50 mM Tris Cl pH 8 0 Triton X 100 solution 20 w v 3 g Triton X 100 12 ml H O Warm in 37 C water bath to dissolve Triton X 100 Store at 4 C stable 2 weeks Urea 8 M 0 75 g ultrapure urea 1 0 ml H O Prepare immediately before use Avoid heating above room temperature Current Protocols in Cell Biology Electrophoresis and Immunoblotting 6 4 29 Supplement 4 Two Dimensional Gel Electrophoresis 6 4 30 Supplement 4 COMMENTARY Background Information Two dimensional gel electrophoresis using isoelectric focusing followed by SDS PAGE is the single most powerful analytical method cur rently available for separating complex protein mixtures such as whole cell or tissue extracts It is therefore a valuable
259. l H20 7 03 ml 7 78 ml Glycerol 4 00 g 3 17 ml 10 w v ammonium persulfate 50 ul 25 ul TEMED 10 ul 5 ul Prepare separating and stacking gel solutions separately In a 50 ml side arm flask mix 30 acrylamide 0 8 bisacrylamide solution Table 6 1 1 Tris Cl SDS pH 8 45 see reagents below and H20 Add glycerol to separating gel only Degas under vacuum 10 to 15 min Add 10 ammonium persulfate and TEMED Swirl gently to mix use immediately ADDITIONAL REAGENTS USED IN GELS Tris Cl SDS pH 8 45 3 0 M Tris Cl containing 0 3 w v SDS Dissolve 182 g Tris base in 300 ml H20 Adjust to pH 8 45 with 1 N HCl Add H20 to 500 ml total volume Filter the solution through a 0 45 um filter add 1 5 g SDS and store at 4 C up to 1 month The recipes produce 30 ml of separating gel and 12 5 ml of stacking gel which are adequate for two gels of dimensions 0 75 mm x 14cm x 14 cm The recipes are based on the Tris tricine buffer system of Schagger and von Jagow 1987 Pall reagents and solutions used in the protocol must be prepared with Milli Q purified water or equivalent Best to prepare fresh Failure to form a firm gel usually indicates a problem with the persulfate TEMED or both Additional Materials also see Basic Protocol 1 Separating and stacking gel solutions Table 6 1 5 2x tricine sample buffer see recipe Peptide molecular weight standards Table 6 1 6 Cathode buffer see recipe Anode buffer see recipe Coomassie blue G 25
260. l applications transferring one gel at a time is recommended The gel next to the anode tends to be more efficiently transferred when blotting more than one gel at a time Transfer proteins from gel to membrane 8 Place top electrode onto transfer stack Most units have safety interlock features and can only be assembled one way Consult manufacturer s instructions for details Once assembled do not move the top electrode This can shift the transfer stack and move the gel relative to the membrane Some transfer will occur as soon as the gel contacts the membrane and any shifting of the transfer stack after assembly will distort the transfer pattern 9 Carefully connect high voltage leads to the power supply see unrr 6 1 for safety precautions Apply constant current to initiate protein transfer Transfers of 1 hr are generally sufficient In general do not exceed 0 8 mA cm of gel area For a typical minigel 8 x 10 cm and standard sized gel 14 x 14 cm this means 60 and 200 mA respectively Monitor the temperature of the transfer unit directly above the gel by touch The unit should not exceed 45 C If the outside of the unit is warm too much current is being applied Note that units with graphite electrodes are more prone to heating because graphite has much more resistance to current flow than platinum or steel electrodes 10 After transfer turn off power supply and disassemble unit Remove membrane from transfer stack
261. le 6 9 1 Once visualization has occurred image capture consists of the following steps previewing the image while Electrophoresis and Immunoblotting 6 9 5 Supplement 16 Digital Electrophoresis Analysis 6 9 6 Supplement 16 Table 6 9 1 Methods Compatability of Popular Image Capture Devices with Common Visualization Image capture device be Pe ae Silver halide CCD Desktop Storage Fluorescent Visualization method photography camera scanner phosphor scanner Optical density Fluorescence Chemiluminescence Radioactivity The device with the highest sensitivity and greatest dynamic range for a visualization method is marked with a other devices that can detect this visualization method are indicated with a and devices that are not suitable for a visualization method are indicated with a A indicates that only some devices of this type can be used with this visualization method Optical density methods include Coomassie blue staining adjusting capture parameters capturing the im age and saving the image for later analysis During the preview process capture pa rameters are optimized for data content and for ease and rapidity of later processing steps Typi cally the first step is to place the sample so that when the image is captured the rectangular edges of the gel are horizontal and vertical on the monitor and any lan
262. le fractions are used the required time can be substantially longer However if utilizing desalting columns to obtain samples with low cation concen trations the time can be shorter Since the sample should be separated immediately by BN PAGE and cannot be frozen and stored one should reserve some time for gel load ing which usually takes another 0 5 to 1 hr Depending on the gel size running of the BN PAGE takes from 6 hr to overnight Loading of two second dimension SDS PAGE gels takes 1to 1 5 hr Running the SDS PAGE takes be tween 1 5 hr and overnight again depending on the size of the gels Thus two dimensional BN SDS PAGE takes at least 1 5 days not counting the visualization process of the pro teins of interest One rate limiting step is the running of the second dimension gels 30 to 40 lanes can eas ily be separated in parallel on two BN gels but loading running and detection from 30 to 40 second dimension gels requires substantial operator time and electrophoresis equipment Therefore whenever possible one should try to use only first dimension BN gels without the need for the second dimension Pouring gradient gels is another time consuming process To minimize the time re quirement it is strongly recommended to use multicasting equipment to pour several gels at once This also ensures best reproducibility for critical comparisons of multiple samples Acknowledgements I thank Michael Reth and Klaus P
263. lfate TEMED 0 02 0 02 0 02 0 02 0 02 0 02 0 02 Preparation of gel In a 75 ml side arm flask mix 30 acrylamide 0 8 bisacrylamide solution see Table 6 1 1 4x Tris gel buffer see Reagents and Solutions and H O If desired degas under vacuum 5 min to speed polymerization Add 10 ammo nium persulfate and TEMED Swirl gently to mix Use immediately The recipes produce 40 ml gel solution which is adequate for one gel of dimensions 1 5 mm x 14 cm x 16 cm or two gels of dimensions 0 75 mm x 14 cm x 16 cm bA reagents and solutions used in the protocol must be prepared with Milli Q purified water or equivalent Units of numbers in table body are milliliters The desired percentage of acrylamide in the gel solution depends on the molecular size of the protein being separated dMust be freshly made e Added just before polymerization 7 Fill wells with electrophoresis buffer If desired prerun gel The gel can be prerun at this point to remove any charged material such as ammonium persulfate from the gel prior to loading the sample Assemble the electrophoresis unit and fill the buffer chambers with electrophoresis buffer Run the gel at 300 V until the current no longer drops This should take 30 min Disassemble the unit discard the buffer and proceed to the next step 8 Carefully load up to 10 ul 0 75 mm gels or 20 ul 1 5 mm gels sample per lane as a thin layer at the bottom of the wells Load control
264. ligosaccharides Anal Biochem 283 136 145 Packer N Pawlak A Kett W Gooley A Red mond J and Williams K 1997 Proteome analysis of glycoforms A review of strategies for the microcharacterization of glycoproteins sepa rated by two dimensional polyacrylamide gel electrophoresis Electrophoresis 18 452 460 Packer N Ball M and Devine P 1999 Glycobi ology and proteomics In 2 D Proteome Analysis Protocols Methods in Molecular Biology A Link ed vol 112 pp 341 352 Humana Press Totowa NJ Patton W 2000a A thousand Points of light The application of fluorescence detection technolo gies to two dimensional gel electrophoresis and proteomics Electrophoresis 21 1123 1144 Patton W 2000b Making blind robots see The synergy between fluorescent dyes and imaging devices in automated proteomics BioTechniques 28 944 957 Raju T 2000 Electrophoretic methods for the analysis of N linked oligosaccharides Anal Biochem 283 125 132 Reuter G and Gabius H 1999 Eukaryotic glyco sylation Whim of nature or multipurpose tool Cell Mol Life Sci 55 368 422 Steinberg T Pretty On Top K Berggren K Kem per C Jones L Diwu Z Haugland R and Patton W 2001 Rapid and simple single nanogram detection of glycoproteins in polyacrylamide gels and on electroblots Pro teomics 1 841 855 Taverna M Tran N Merry T Horvath E and Ferrier D 1998 Electrophoretic methods
265. linical appli cation of a rapid method using agarose gel elec trophoresis and western blotting to evaluate von Willebrand factor protease activity Electropho resis 22 946 949 Peacock A C and Dingman W C 1968 Molecular weight estimation and separation of ribonucleic acid by electrophoresis in agarose acrylamide composite gels Biochemistry 7 668 674 Rick M R 2002 Hemophilia and von Willebrand disease In UpToDate Clinical Reference Li brary Release 9 1 B D Rose ed UpToDate Wellesley MA Shainoff J R 1993 Electrophoresis and direct im munoprobing on glyoxal agarose In Advances Current Protocols in Cell Biology in Electrophoresis Vol 6 A Chrambach M J Dunn and B J Radola eds pp 65 176 VCH Publishers New York Shainoff J R Urbanic D A and DiBello P M 1991 Immunoelectrophoretic characterizations of the cross linking of fibrinogen and fibrin by factor XIa and tissue transglutaminase Identi fication of a rapid mode of hybrid alpha gamma chain cross linking that is promoted by the gamma chain cross linking J Biol Chem 266 6429 6437 Key References Krizek and Rick 2000 See above This original paper of the procedure and use of immunoblotting and chemiluminescence for ararose gel electrophoresis provides the background and reasons for the development of this assay in the clinical laboratory setting Hoyer and Shainoff 1980 See above This paper outlines the original
266. ll accommodate the first dimension gel Narrow analytical isoelectric focusing gels lt 1 5 mm that fit between the glass plates of the second dimension gel do not generally require a stacking gel although a stacking gel may improve resolution under some circumstances Stacking gels are essential when first dimen sion gels gt 1 5 mm are loaded on reduced thickness second dimension gels for example when 3 mm first dimension gels are loaded on 1 5 mm second dimension gels To ensure the best reproducibility casting multiple second dimension gels in a multigel casting stand is strongly recommended This is especially important when gradient gels are used for the second dimension and or critical comparisons of multiple samples are planned This protocol describes all the specific steps required for successfully casting and running the second dimension gel The use of beveled plates and an agarose overlay is especially important when 3 mm IEF gels are loaded onto 1 5 mm second dimension gels Materials 2 w v agarose see recipe Equilibration buffer see recipe Isoelectric focusing gels containing protein samples to be analyzed see Basic Protocol 1 Piece of agarose containing molecular weight standards see Support Protocol 6 Beveled glass plates Boiling water bath Metal or plastic scoop Additional reagents and equipment for linear and gradient Laemmli gels UMIT 6 1 Current Protocols in Cell Biology agarose overlay IE
267. ll as 5 kDa Current Protocols in Cell Biology Additional Materials also see Basic Protocol 1 Separating and stacking gel solutions Table 6 1 7 2x Tris Cl SDS pH 8 8 dilute 4x Tris Cl SDS pH 8 8 Table 6 1 1 2x SDS electrophoresis buffer see recipe 1 Prepare and pour the separating and stacking gels see Basic Protocol 1 steps 1 to 11 using the modified recipes in Table 6 1 7 After removing the isobutyl alcohol overlay from the separating gel rinse with 2x Tris Cl SDS pH 8 8 rather than 1 x Tris CI SDS 2 Prepare the sample and load the gel see Basic Protocol 1 steps 12 to 20 but substitute 2x SDS electrophoresis buffer for the 1x SDS electrophoresis buffer Table 6 1 6 lists the standards for small protein separations Table 6 1 7 Recipes for Modified Laemmli Peptide Separating and Stacking Gels SEPARATING AND STACKING GELS Stock solution Separating gel Stacking gel 30 w v acrylamide 0 8 w v 10 00 ml 0 65 ml bisacrylamide 8x Tris Cl pH 8 8 3 75 ml 4x Tris Cl pH 6 8 1 25 ml 10 w v SDS 0 15 ml 50 ul H20 1 00 ml 3 00 ml 10 w v ammonium persulfate 50 ul 25 ul TEMED 10 ul 5 ul Prepare separating and stacking gel solutions separately In a 25 ml side arm flask mix 30 acrylamide 0 8 bisacrylamide solution see Table 6 1 1 8x Tris Cl pH 8 8 or 4x Tris Cl pH 6 8 see reagents below 10 SDS and H20 Degas under vacuum 10 to 15 min Add 10 ammonium persulfa
268. llow the plug solution to push the acrylamide in the caster up into the plates Close the outlet when the plug solution reaches the bottom of the plates A discontinuity between the bottom of the gels and the plug solution will be obvious 9 Quickly add 100 ul H2O saturated isobutyl alcohol to each gel sandwich Let the gels polymerize undisturbed for 1 hr 10 Prepare and pour the stacking gel see Basic Protocol 2 steps 9 and 10 Disassemble the system 11 Disconnect the gradient maker place the caster in a sink and remove the front faceplate The plug solution will drain out from the bottom of the caster 12 Remove the gels see Basic Protocol 2 step 11 Gradient minigels can be stored as described for single concentration minigels see Basic Protocol 2 step 11 annotation For instructions on preparing loading and running the gels see Basic Protocol 2 steps 12 to 17 Current Protocols in Cell Biology Electrophoresis and Immunoblotting 6 1 27 Supplement 37 SUPPORT CALCULATING MOLECULAR MASS EGE Determining the molecular mass of an unknown protein or nucleic acid fragment is straightforward given the use of calibration size standards in the same gel Typically a tracking dye such as bromphenol blue is added to the sample prior to loading on the gel e g see recipe for SDS sample buffer The tracking dye moves ahead of the proteins and serves as a relative mobility marker A set of protein standards
269. llow transfer of very high molecular weight proteins Thorough washing after blocking buffer and antibody additions is important in both protocols See Table 6 7 1 for trou bleshooting agarose gel electrophoresis and immunoblotting Anticipated Results The radiographs that result from the chemi luminescent and radioactive detection proce dures show a wide distribution of multimer sizes of normal von Willebrand factor In cer tain subtypes of von Willebrand disease i e Type 2 there is a marked or modest decrease in the higher molecular weight multimers Fig 6 7 2 If newly synthesized von Willebrand factor is extracted from platelets the unusually high molecular weight multimers are seen Fig 6 7 3 In samples that are incubated under conditions that activate the von Willebrand fac tor protease a decrease in the high and inter mediate sized multimers is seen Fig 6 7 4 Rick and Krizek unpub observ Time Considerations Both protocols should be started in the morning to allow sufficient time for electropho resis Horizontal electrophoresis and blotting can be completed within 48 hours blotting is conveniently completed overnight and detec Current Protocols in Cell Biology 900 kDa gt 700 kDa gt PNP 78 Hebel amp Do gs s 53 79 Figure 6 7 4 Luminograph of pooled normal plasma PNP showing normal vWF multimers lane 1 and proteolysed multimers of normal vWF from PNP afte
270. log protein mass on the y axis versus relative mobility of the standards on the x axis Fig 6 1 6 3 Perform linear regression using a calculator or analysis program 4 Use the linear regression equation y mx b to estimate the mass of the unknown Log molecular weight slope mobility of unknown y intercept A T a O _ amp i oO O 0 0 0 2 0 4 0 6 0 8 1 0 1 2 Rf B 300000 200000 e T a 90000 80000 70000 60000 S 50000 8 40000 gt 30000 20000 g 0 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 Rf Figure 6 1 6 Standard protein molecular weight curves for A single concentration 5 and 12 5 and B gradient 5 to 20 gels Protein standards are separated via SDS PAGE visualized by staining with Coomassie blue UNIT 6 6 and measured relative to the dye front to give the relative mobility R Note the single concentration gel has a more limited range of linearity than the gradient gel The standard curve permits the calculation of the molecular weight of an unknown by using the R of the unknown to predict the molecular weight Current Protocols in Cell Biology Electrophoresis and Immunoblotting 6 1 29 Supplement 37 One Dimensional SDS PAGE 6 1 30 Supplement 37 REAGENTS AND SOLUTIONS Use Milli Q water in all recipes and protocol steps For common stock solutions see APPENDIX 2A for suppliers see SUPPLIERS APPENDIX Anode buffe
271. ls for commonly used vertical minigel and standard sized SDS PAGE appa ratuses are available from several manufactur ers Table 6 1 4 Flatbed horizontal isoelec tric focusing IEF and SDS PAGE gels are not listed Hoefer supplies a range of horizontal gels for a variety of applications and should be consulted for further information When using precast gels pay strict attention to shelf life In general manufacturers overrate the shelf life and the sooner the gels are used the better When reasonably fresh precast gels provide excellent resolution that is as good or better than a typical gel cast in the laboratory The most widely used method for discon tinuous gel electrophoresis is the system de scribed by Laemmli 1970 This is the de naturing SDS discontinuous method used in Basic Protocol 1 A discontinuous buffer sys tem uses buffers of different pH and com position to generate a discontinuous pH and voltage gradient in the gel Because the dis continuous gel system concentrates the pro teins in each sample into narrow bands the applied sample may be more dilute than that used for continuous electrophoresis In the discontinuous system the sample first passes through a stacking gel which has large pores The stacking gel buffer contains chloride ions called the leading ions whose electrophoretic mobility is greater than the mobility of the proteins in the sample The electrophoresis buffer contains glycine ion
272. lso be applied to a lane or two Typically 2 ul of this standard is diluted in 6 ul of sample buffer and heated in the same manner as the samples to be characterized These standards contain a mixture of glycosylated and non glycosylated proteins ranging from 14 to 180 kDa in molecular weight The standards serve as molecular weight markers and as alternate bands of positive and negative controls for glycoprotein and total protein detection Each protein is present at 0 5 mg ml 2 Separate proteins by SDS polyacrylamide gel electrophoresis using standard meth ods UNIT 6 1 The procedure is optimized for gels that are 0 5 to 1 mm thick Prepare blot 3 After electrophoresis transfer the proteins to PVDF membrane using standard electroblotting procedures UNIT 6 2 The use of nitrocellulose membranes is not recommended 4 After transfer fix the blot by immersing in 25 ml fix solution and incubate with gentle agitation e g on an orbital shaker at 50 rpm for 45 min at room temperature 5 Wash the blot by incubating in 25 ml wash solution with gentle agitation for 10 min room temperature Repeat this wash step one additional time 6 Oxidize the blot in 25 ml periodic acid solution with gentle agitation for 30 min 7 Wash the blot in 25 ml wash solution with gentle agitation for 5 to 10 min Repeat this washing step two additional times Visualize glycoproteins 8 Prepare fresh Pro Q Emerald 300 staining solution by diluting
273. ly not the complete dynamic range of the system An additional considera tion is the dynamic range of the visualization method Many popular visualization methods have linear dynamic ranges of 1 to 2 5 orders of magnitude An imaging system with greater dynamic range analyzing the results of such a visualization method will not improve the dy namic range Saturation Saturation occurs when a detector or visu alization method receives input levels beyond Current Protocols in Cell Biology A normal 100 5 2E i mn Lot 2 3 1 Input O 100 200 300 400 500 EH GHA B contrast 100 5 2 i s Hadal OO il 0 I Input O 100 200 300 400 500 IIT TT brightness 100 9 S 2 i O a 0 Input 0 100 200 300 400 500 IlI l D amma 100 9 5 2g el Si COS 0 l I Input O 100 200 300 400 500 CE E Bei 1s E saturation 100 0 O 100 200 300 400 500 Eta E Tif Figure 6 9 1 Examples of how altering image capture settings affects the image and the analysis The graph on the left displays the light intensity response curve used for image capture while the image and resulting lane profile on the right display how the setting affects the image The lane profile displays pixel position versus normalized pixel intensity A In this case the output has not been altered giving a straight line with a slope of 1 on the response curve B The im
274. ly on Laemmli based discontinuous SDS PAGE but has the expected mobility after electrophoresis in the phosphate based continuous system de scribed here This is also true of cross linked proteins Multiple gel casting Support Protocols 1 to 3 is appropriate when gel to gel consis tency is paramount or when the number of gels processed exceeds five a week The va riety of multiple gel casters gradient makers and inexpensive pumps available from major suppliers simplifies the process of casting gels in the laboratory Alternatively precast gradi ent gels are available for most major brands of gel apparatuses Table 6 1 4 Minigels Basic Protocol 2 are generally considered to be in the 8 x 10 cm size range Current Protocols in Cell Biology although there is considerable variation in ex act size Every technique that is used on larger systems can be translated with little difficulty into the minigel format This includes stan dard and gradient SDS PAGE and separations for immunoblotting and peptide sequencing Two dimensional SDS PAGE electrophoresis also adapts well but here the limitation of separation area becomes apparent for high resolution separations large format gels are required Gradient minigels Support Proto col3 are popular due to the combination of separation range and resolution Matsudaira and Burgess 1978 They are particularly use ful for separation of proteins prior to peptide sequencing Myla
275. lysis by mass spectrometry One Dimensional SDS PAGE 6 1 8 Supplement 37 Prepare the sample and load the gel 12 Dilute a portion of the protein sample to be analyzed 1 1 v v with 2x SDS sample buffer and heat 3 to 5 min at 100 C in a sealed screw cap microcentrifuge tube If the sample is a precipitated protein pellet dissolve the protein in 50 to 100 ul of 1x SDS sample buffer and boil 3 to 5 min at 100 C Dissolve protein molecular weight standards in 1x SDS sample buffer according to supplier s instructions use these standards as a control Tables 6 1 2 and 6 1 3 For dilute protein solutions consider using 5 1 protein solution 6 x SDS sample buffer to increase the amount of protein loaded Proteins can also be concentrated by precipitation in acetone ethanol or trichloroacetic acid TCA but losses will occur For a0 8 cm wide well 25 to 50 ug total protein in lt 20 ul is recommended for a complex mixture when staining with Coomassie blue and 1 to 10 ug total protein is needed for samples containing one or a few proteins If silver staining is used 10 to 100 fold less protein can be applied 0 01 to 5 ug in lt 20 ul depending on sample complexity To achieve the highest resolution possible the following precautions are recommended Prior to adding the sample buffer keep samples at 0 C Add the SDS sample buffer room temperature directly to the 0 C sample still on ice in a screw top microcentrifuge
276. m cell Bio Rad or SE 600 400 16 cm unit Hoefer with clamps glass plates casting stand and buffer chambers 0 75 mm spacers 0 45 um filters used in stock solution preparation 25 ml Erlenmeyer side arm flasks Vacuum pump with cold trap 0 75 mm Teflon comb with 1 3 5 10 15 or 20 teeth Screw top microcentrifuge tubes recommended 25 or 100 ul syringe with flat tipped needle Constant current power supply see Electricity and Electrophoresis above Pour the separating gel 1 Assemble the glass plate sandwich of the electrophoresis apparatus according to manufacturer s instructions using two clean glass plates and two 0 75 mm spacers If needed clean the glass plates in liquid Alconox or RBS 35 Pierce These aqueous based solutions are compatible with silver and Coomassie blue staining procedures 2 Lock the sandwich to the casting stand 3 Prepare the separating gel solution as directed in Table 6 1 1 degassing using a rubber stoppered 25 ml Erlenmeyer side arm flask connected with vacuum tubing to a vacuum pump with a cold trap After adding the specified amount of 10 ammonium persulfate and TEMED to the degassed solution stir gently to mix The desired percentage of acrylamide in the separating gel depends on the molecular size of the protein being separated Generally use 5 gels for SDS denatured proteins of 60 to 200 kDa 10 gels for SDS denatured proteins of 16 to 70 kDa and 15 gels for SDS denatured pr
277. mag nitude compared to 1 5 for film Johnston et al 1990 This makes it possible to accurately Electrophoresis and Immunoblotting 6 3 9 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots 6 3 10 Table 6 3 2 Film Choice and Exposure Temperature for Autoradiography Isotope Enhancement method Film Exposure temperature 3H Fluorography Double coated 70 C 35S 14C 3P None Single coated Room temperature 358 14C 3P Fluorography Double coated 70 C 32p 125 CaWO intensifying screens Double coated 70 C quantitate very weak or very strong radioactive Anticipated Results samples Troubleshooting Cracking is one of the most common prob lems encountered when drying gels This often occurs if the gel is removed from the dryer before it has adequately dried or if drying tem peratures are too high To overcome this prob lem drying times should be extended and the performance of the vacuum pump and heater unit should be checked For many gels particu larly for those with high percentages of poly acrylamide or gt 1 5 mm thick cracking can be reduced by using an alternative fixing solution containing glycerol 3 glycerol 10 glacial acetic acid 20 methanol see Support Proto col 1 Among the biggest problems encountered in autoradiography are images that are either too weak or too intense Such problems can be solved by varying the exposure time Estimat ing initial ex
278. menon of the photographic process and dif ferences in reduction potential of silver at the sites occupied by macromolecules relative to that at the rest of the gel matrix The differences in reduction potentials can be manipulated to obtain either a positive or negative image of protein bands Positive silver staining protocols comprise the following stages 1 fixation of proteins and elimination of interfering sub stances e g amino acids Tris SDS 2 sen sitization with an agent to increase sensitivity and or contrast 3 impregnation with silver solution silver nitrate basic silver ammonia or silver diamine complex 4 controlled rinse to remove silver ions not associated with proteins 5 image development and 6 image devel opment stopping reviewed by Rabilloud et al 1994 The procedure described in Basic Pro tocol 2 Blum et al 1987 involves sensitiza tion with thiosulfate impregnation with silver nitrate and image development using a dilute formaldehyde solution at high pH Small amounts of thiosulfate included in the devel oper solution help to keep the background low by complexing silver ions that otherwise could form spurious silver deposits in the gel matrix This procedure is highly sensitive and compat ible with virtually all types of PAGE systems Unlike other silver staining protocols which yield a mixture of positive and negative bands or different band colors the images of the protein bands
279. mensional analysis software opti mized for lane based band detection Images from two dimensional electrophoresis are best handled by specific programs designed to de tect spots and to assign two mobility values and a quantity value to the spot After the initial characterization of bands and spots compari sons are often made between bands or spots from different experiments through the use of database programs and matching algorithms Software for One Dimensional Analysis Lane positioning For one dimensional analysis the first ac tivity is to detect the lanes on the image One of three different methods is commonly em ployed for this For images with straight well defined lanes with a large number of bands automatic lane detection algorithms can quickly and accurately place the lanes On im ages with very well defined lanes such as pseudoimages from finish line type electro phoresis equipment automated lane calling based on image position is possible For images with smiling bent or irregular lanes manual positioning of the lanes is often the fastest and most accurate method of lane definition Re gardless of the method of identifying the lanes the lane boundaries need to be carefully set for accurate quantitation and mass determinations Lane widths should be wide enough so that the entire area of all bands in that lane are included but they should not be so wide as to include bands from adjacent lanes To acco
280. method for following dis ease related changes or for detecting changes in protein expression under diverse experimental conditions To achieve maximum reproducibility between samples to be compared multiple gels should be cast and run simultaneously Anderson and Anderson 1978a b Despite the exceptionally high resolving power of the method the total number of proteins that can be resolved in a single two dimensional gel is only 1000 to 2000 whereas the total number of proteins in a single mammalian cell type is likely to be at least 10 to 20 times higher Therefore the old guideline that a single spot on a two dimensional gel is a single protein needs to be revised as analytical detection methods im prove Conversely a single protein single gene product can produce multiple usually adjacent spots in the isoelectric focusing dimension owing to variable degrees of chemical or posttransla tional modification Common examples of vari able posttranslational modifications that can usu ally be detected on two dimensional gels include phosphorylation glycosylation and acetylation Examples of chemical modifications that cause charge heterogeneity include deamidation of side chain amines usually asparagines oxida tion of sensitive side chains and modification of lysines Potential modification of lysines by urea is particularly important because urea rapidly decomposes to form cyanate which readily reacts with amino groups espe
281. mit steps 12 to 19 do not prefocus the gels 20 Remove the 8 M urea polymerization overlay solution and place 50 ul lysis buffer on top of each gel Wait at least 2 min then remove the lysis buffer 23 Carefully fill all tubes with the upper chamber electrode anode solution prepared by mixing pH 2 11 ampholytes with water in a 1 40 ratio 24 Fill the upper buffer chamber anode with the solution described in step 23 Iminodiacetic acid 10 mM may be a more economical alternative anode solution 26 Focus for a total of 4000 Vhr NONEQUILIBRIUM ISOELECTRIC FOCUSING OF BASIC PROTEINS In general most equilibrium IEF gel systems using soluble ampholytes produce pH gradients that do not exceed pH 8 0 on the basic end yet many proteins have higher pI values For this reason samples containing very basic proteins are usually focused using a nonequilibrium system In an equilibrium system proteins are loaded on the basic end of the gel and migrate toward the acidic end until they reach a pH equal to their pI In nonequilibrium systems the sample is loaded on the acidic end of the gel and focusing is terminated after a relatively short time fewer volt hours To run nonequilibrium IEF gels follow the procedure previously described see Basic Protocol 1 with these alterations in the indicated steps Current Protocols in Cell Biology 8 Use 0 1 M NaOH as the lower electrode solution Electrode solutions and electrodes are re
282. mmediately fixed with isopropanol glacial acetic acid fixing solution After the gel is washed it is blocked with ethanolamine BSA The gel after a second series of washes is incubated with I labeled anti vWF antibody for 10 to 24 hours at room temperature After extensive washing and drying the gel is placed in a cassette with film and exposed 1 to 5 days Materials In gel sample buffer fresh see recipe Borate saline buffer BSB see recipe Sample 0 5 w v bromphenol blue in H O Isopropanol Agarose gel buffer see recipe SeaKem HGT P agarose FMC BioWhittaker Molecular Agarose running buffer see recipe Fixing buffer see recipe Blocking buffer in gel see recipe 25T labeled rabbit anti human vWF polyclonal antibody Dako A0082 radiolabel using protocol of choice and immunopurify Hoyer and Shainoff 1980 also see UNIT 7 10 2 w v human IgG see recipe High salt wash buffer see recipe 12 x 75 mm polypropylene tube 12 5 x 26 0 x 0 3 cm glass plate Amersham Pharamacia Biotech 12 5 x 24 0 cm spacer plate with adherent 0 5 mm spacers Amersham Pharmacia Biotech 12 4 x 25 8 cm GelBond film Amersham Pharmacia Biotech Flexiclamps Amersham Pharmacia Biotech 20 ml syringe 60 C oven Aluminum foil Gelman Delux electrophoresis chamber Gelman Sciences or equivalent Current Protocols in Cell Biology 104 x 253 mm paper electrophoresis electrode wicks Amersham Pharmacia Biotech
283. mmended The gradient gels can be stored for several days at 0 to 4 C before casting the stacking gel Time Considerations Preparation of separating and stacking gels requires 2 to 3 hr Gradient gels generally take 5 min to cast singly Casting multiple single concentration gels requires an addi tional 10 min for assembly Casting multiple gradient gels takes 15 to 20 min plus assembly time It takes 4 to 5 hr to run a 14 x 14 cm 0 75 mm gel at 15 mA 70 to 150 V and 3 to 4 hr to run a 0 75 mm gel at 20 mA 80 to 200 V Overnight separations of 12 hr require 4 mA per 0 75 mm gel It takes 4 to 5 hr to run a 1 5 mm gel at 30 mA Electrophoresis is normally performed at 15 to 20 C with the temperature held constant using a circulat ing water bath For air cooled electrophoresis units lower currents and thus longer run times are recommended It takes 1 hr to run a 0 75 mm minigel at 20 mA 100 to 120 V Separation times are not significantly different for gradient minigels Literature Cited Dhugga K S Waines J G and Leonard R T 1988 Correlated induction of nitrate uptake and membrane polypeptides in corn roots Plant Physiol 87 120 125 Gallagher S R and Leonard R T 1987 Elec trophoretic characterization of a detergent treated plasma membrane fraction from corn roots Plant Physiol 83 265 271 Hunkapiller M W Lujan E Ostrander F and Hood L E 1983 Isolation of microgram quan tities of p
284. mount of Tris SDS buffer in standards as in experimental samples Prepare labeled cryovials to store aliquots of the sample if desired Precool vials on ice prior to making aliquots The amount of protein per aliquot depends on the anticipated future uses of the sample as repeated freezing and thawing should be avoided About 500 ug gel whole cell extract is a maximum load for preparative purposes using 3 mm gels i e isolation of proteins for sequencing or other structural work Approximately 50 ug gel is an appropriate load for silver staining The final protein concentration after completion of the protocol steps 15 to 20 will equal the concentration found by protein assay divided by 1 1 owing to the addition of reagents after the protein assay step Add 20 ul DNase and RNase solution per 400 ul Tris SDS buffer used for sonication step 11 Incubate 10 min on ice Add 20 ul of 20 SDS solution and 5 ul of 2 mercaptoethanol per 400 ul Tris SDS buffer used in step 11 Incubate 5 min at 37 C Quickly divide samples into previously prepared cryovials and immediately freeze aliquots using a dry ice ethanol bath Store at 80 C Samples stored at 80 C are stable 21 year Work quickly to minimize potential proteolysis This step may be omitted if the samples are to be loaded on IEF gels immediately Generally the total amount of sample greatly exceeds the amount required for an IEF gel and freezing aliquots is beneficial To avo
285. mple volumes between 5 and 20 ul make sure contact between the strip and the gel is uniform Use sample application pieces for sample volumes gt 20 ul Remove the application pieces halfway through focusing For sample volumes of 2 to 10 ul samples may be spotted directly on the gel without using applicator strips See manufacturer s instructions for further details An important experimental consideration is the position in the pH gradient where the sample is applied The acidic end of the gel can usually be used for sample application however the optimal loading position may need to be determined empirically for different types of samples At high protein concentrations and or at nonoptimal pHs samples may precipitate in the gel at the loading position Current Protocols in Cell Biology Electrophoresis and Immunoblotting 6 4 15 Supplement 4 SUPPORT PROTOCOL 3 Two Dimensional Gel Electrophoresis 6 4 16 Supplement 4 Samples should contain lt 50 mM salt or buffer components greater concentrations will cause local overheating of the gel If possible salt free samples should be solubilized or dialyzed in the rehydration buffer 15 Align the electrodes with the electrode strips put the safety lid in place and connect the apparatus to the power supply Conduct electrophoresis at 3000 V Broad range IPG gels such as Immobiline DryPlate pH 3 10 should be run at 3000 V for 2 to 4 hr Narrower range g
286. mplex 4 Load the BN gel at 4 C as in Basic Protocol 1 Load the samples in the order of the dilution see Fig 6 10 2 At the end also load the solution containing the multiprotein complex with no antibody as reference Do not load any sample in the well next to the sample with the highest concentration of antibody In this well the marker mix can be loaded If a second series of antibody added samples is loaded use the reverse order see Fig 6 10 2 Run and analyze the Blue Native gel 5 Run NAMOS BN gels as described in the Basic Protocol 1 except reduce the voltage at the beginning of the separation if a small gel has been prepared e g using BioRad Protean II or III run at 50 V if a large gel has been prepared e g using BioRad Protean II xi run at 75 Continue electrophoresis at the appropriate abovementioned voltage until the sample has entered the separating gel At that point increase voltage to 180 V for small gel or 400 V for large gel 6 Analyze the gels by immunoblotting Alternate Protocol 1 or 2 using only antibodies that recognize the protein protein complex of interest The detection method should not stain the monoclonal antibodies used at steps I and 2 For example for detection use directly labeled antibodies or polyclonal rabbit antiserum in combination with labeled anti rabbit IgG antibodies that do not cross react with the antibodies that were used for the shift In order to control for the cross re
287. mplish this curved or bent lanes might need to be used in order to follow the electrophoresis lane pattern Lane length and position also must be adjusted as necessary so that all bands of interest are included If mass determinations are necessary the sample loading point should probably also be included in the lane or be the start of the lane At this point lines of equal mobility often called R or iso molecular weight lines are added to the image as necessary These lines allow for correction of lane to lane deviations in the mobility of reference bands and generate more accurate measurements of mass A similar form of correction is also possible for within lane correction of mobilities This correction is important for accurate detection and quantita tion of closely spaced bands Band detection Once the lanes have been defined the bands present in each lane need to be detected There are many methods for detecting bands One method is to systematically scan the lane profile from one end to the other identifying regions of local maxima as bands Another common method is to use first and second order deriva tives of the lane image or lane profile in order to find inflection points in the change of slope in pixel intensity values Patton 1995 Regard less of the method used it is often necessary to alter the search parameters so that they perform reliably under a given experimental condition Current Protocols in Cell Bi
288. n Lossy compression is sometimes necessary for applications with ex tremely large image files such as real time video capture but it usually represents an unaccept able loss of data if used with electrophoresis image capture Many different file types have been devel oped to store digital images Some of these file types are proprietary or hardware specific For example PICT is a Macintosh format and BMP is a PC compatible format Each file type has its own structure Some types do not allow compression for others it is optional and for some it is mandatory File types vary in the types of images they support particularly in the number of colors or gray levels Below is a brief description of a few of the more prevalent file types TIFF Tagged Image File Format is one of the most commonly used formats It is particu larly versatile since it is an open format that can be modified for specific applications One rea son for its versatility is the ability to attach or tag data to the image The tags can include information such as optical density calibration resolution experimenter date of capture and any other data that the application software supports TIFF images can be monochrome 4 8 or 16 bit gray scale or one of many color image formats Compression is optional with LZW RLE and JPEG often supported Russ 1995 Since TIFF is supported by both Macin tosh and PC computers it is a good choice for multiple platfo
289. n e g goat horse or rabbit normal serum from the same species as the primary antibody can reduce the back ground presumably by reducing cross reactiv ity between the primary antibodies and the blocking agent Combinations of blocking agents can also be effective Thus 0 1 human serum albumin HSA and 0 05 Tween 20 in TBS is recommended when probing Immo bilon P membranes with human serum Craig et al 1993 However this can also lead to overall loss of antigen signal requiring a ten fold increase in the primary antibody serum concentration to achieve an adequate back ground free antigen signal When using chemiluminescent detection for immunoblotting high background frequently occurs particularly for strong signals Pampori et al 1995 Several methods are available for reducing the background from chemilumines cent reactions These include changing the type and concentration of blocking agents see above optimizing antibody concentrations letting the reaction proceed for several minutes before exposing to film or simply limiting the exposure time of the film on the blot These procedures are not always successful however and can lead to inconsistent results An alterna tive approach is to reduce the concentration of reagents ten fold This effectively removes the background and has a number of advantages which include lower cost increased signal to noise ratio and reduced detection of cross re acting spe
290. n a given lane By eluting the dye off the strip and reading in a spectrophotometer 4 an internal control value of protein on a lane is obtained This value is used to correct for any differences in protein loading from lane to lane Comparison of the Ponceau S value to the chemilumi nescent or chromogenic immunodetection value determined by densitometry provides a straightforward correction for lane to lane variation This method works best for complex mixtures where the immunodetected protein represents a small proportion of the total protein Klein et al 1995 Additional Materials also see Basic Protocol 1 Spectrophotometer and 2 ml cuvette Current Protocols in Cell Biology SUPPORT PROTOCOL 1 SUPPORT PROTOCOL 2 Electrophoresis and Immunoblotting 6 2 7 BASIC PROTOCOL 2 Immunoblotting and Immunodetection 6 2 8 1 Following protein transfer to nitrocellulose PVDF or nylon see Basic Protocol 1 or Alternate Protocol 1 stain membrane photograph and destain see Support Proto col 1 Membranes should be destained until the background becomes white 2 Mark lanes with a soft pencil and cut lanes into strips 3 Place each strip into 7 ml of distilled water for 7 min and remove the resulting solution If any particulates are visible centrifuge 30 min at 2000 rpm to remove them 4 Read A525 in a 2 ml cuvette Any variation in gel to gel sample loading and blotting efficiency will be reflected
291. n absorption which then impresses the film The unit contains several protocols for autoradiographic detection of various radionuclides including methods for enhancing the signal with intensifying screens or by fluorography Also included in unit 6 3 are discussions of the quantification of film images by densitometry and the direct detection and quantification of radioactive samples in gels by phosphor imaging The resolution of electrophoretic techniques can be enormously enhanced by combining two different electrophoretic procedures performed successively in perpendicular directions i e two dimensional gel electrophoresis The most common type of two dimensional gel electrophoresis is based on separation of proteins by isoelectric focusing on a tube gel first dimension followed by SDS PAGE on a slab gel second dimension The two processes separate proteins on the basis of charge and size respectively allowing resolution of up to several thousand proteins on a single two dimensional gel uniro 4 describes several methods for separating proteins by two dimensional isoelectric focusing SDS PAGE In addition this unit presents a protocol for two dimensional nonreducing reducing electrophoresis in which proteins are separated by SDS PAGE under nonreducing conditions in the first dimension and under reducing conditions in the second dimension This type of two dimensional gel electrophoresis allows analysis of intersubunit disulfide bonds in multi
292. n and data entry easier since if one spot or band is matched to others and is characterized or anno tated this information is easily passed to all the other matches An underlying assumption of matching is that objects with similar separation properties are actually similar Care must be taken to confirm the identity of matched spots or bands by other methods on critical experi ments A simple form of matching is to link bands or spots at similar positions on the gel images This works well when separation and imaging conditions are uniform This is very seldom the case since slight differences in the electropho resis visualization and imaging conditions across a gel and between gels generates incor rect matching with this method Since bands on one dimensional gels are relatively easy to cali brate for mobility matching can occur along contours of equal mobility This dramatically decreases but does not eliminate the variability in detecting similar bands Much of the remain ing variability can be attributed to calibration errors This error can often be compensated for by allowing a small tolerance in mobility values in determining whether a band is matched or not Because of the difficulties in calibrating mobility in two dimensional gels it is often more practical to use matched spots for cali brating mobility than vice versa Spot matching between two two dimensional images starts with finding a small number of landmark spot
293. n apparatus These protocols describe methods for the separation and identification of von Willebrand factor vWF an extremely large plasma protein that is comprised of multimers ranging from 850 000 to 20 000 000 Da The methods can be applied to other mixtures containing large proteins multimeric proteins and other large protein complexes such as fibrinogen and fibrin complexes Shainoff 1991 While the use of an SDS buffer system allows separation of proteins on the basis of size nondenatured proteins can be separated in native agarose gels if their charge and configuration allow for satisfactory partitioning Von Willebrand factor multimers are stable in SDS due to their disulfide linkages and the protocols below utilize SDS The agarose gel electrophoresis and blotting with immunodetection procedure see Basic Protocol utilizes identification of protein by a specific antibody followed by chemiluminescent detection methods Major advantages of this method include the technical ease of preparing the gel increased sensitivity and a much shorter turn around time than in gel antibody analysis see Alternate Protocol It also eliminates the use of radioactive isotopes In addition the primary and secondary antibodies can be easily removed and the membrane PVDF probed again with a different detection antibody This allows the laboratory the opportunity to detect a second protein or antigenic site on the same protein and also a second c
294. n image Incorrect brightness levels can lead either to high background and potential image saturation or as is illustrated in Figure 6 9 1C to a total loss of background information and partial loss of band information Gamma Nonlinear corrections are often applied to images to compensate for how the eye perceives changes in intensity how display devices re produce images or both The most common correction is an exponential one with the ex ponent in the equation termed the gamma A typical gamma value for camera based systems is 0 45 to 0 50 and is illustrated in Figure 6 9 1D This is a compromise value that com pensates for the 2 2 to 2 5 gamma present in most video monitors and the print dynamics of most printers Since it is a nonlinear correction special care must be taken if quantitation is desired Unless directed to by the manufacturer gamma values other than 1 0 should be avoided when quantitating More information on gamma correction can be found on Poynton s Gamma FAQ www inforamp net poynton Poynton color html Dynamic Range Dynamic range describes the breadth of in tensity values detectable by a system and is usually expressed in logarithmic terms such as orders of magnitude decades or optical density OD units A large dynamic range is important when trying to quantitate over a wide range of concentrations The most accurate quantitation occurs in the linear part of the dynamic range which is usual
295. n standard immunoblotting applications Typically lectin conjugates of biotin along with enzyme conjugates of streptavidin or direct conjugates of lectin and enzyme are utilized along with chromogenic fluorogenic or chemiluminescent substrates Just as in immunoblotting the most popular enzymes used to detect lectin or streptavidin are alkaline phosphatase and horseradish peroxidase This unit describes periodate Schiff s base and lectin methods for the detection of glycoproteins The Pro Q Emerald 300 glycoprotein detection method permits fluores cent direct detection of glycoproteins in gels see Basic Protocol 1 or on blots see Alternate Protocol without the use of enzyme amplification systems The method may also be used to detect lipopolysaccharides constituents of the outer membrane surround ing gram negative bacteria The Pro Q glycoprotein blot stain protocol for concanavalin A see Basic Protocol 2 is suitable for the detection of glycoproteins containing Q mannopyranosyl and o glucopyranosyl residues on blots using an alkaline phos phatase based signal amplification system Using different enzyme lectin conjugates such as alkaline phosphatase conjugates of wheat germ agglutinin or Griffonia simpliifo lia lectin II GS II the method can be adapted to the detection of other glycan structures present in glycoproteins Contributed by Wayne F Patton Current Protocols in Cell Biology 2002 6 8 1 6 8 15 Copyright 2002 by J
296. n these gels the proteins are positively charged because of the very low pH of the gel buffers e g acetic acid urea gels for histone separations or the presence of a cationic detergent e g cetyltrimethyl ammonium bromide CTAB Proteins move toward the negative electrode cathode in cationic gel systems and the polarity is reversed compared to SDS PAGE the red lead from the lower buffer chamber is attached to the black outlet of the power supply and the black lead from the upper buffer chamber is attached to the red outlet of the power supply Most SDS PAGE separations are performed under constant current consult instructions from the manufacturer to set the power supply for constant current operation The resistance of the gel will increase during SDS PAGE in the standard Laemmli system If the current is constant then the voltage will increase during the run as the resistance goes up Power supplies usually have more than one pair of outlets The pairs are connected in parallel with one another internally If more than one gel is connected directly to the outlets of a power supply then these gels are connected in parallel Fig 6 1 1 Ina parallel circuit the voltage is the same across each gel In other words if the power supply reads 100 V then each gel has 100 V across its electrodes The total current however is the sum of the individual currents going through each gel Therefore under constant current it is necessary to
297. nal UNIT6 1 OY UNIT6 4 are visualized as clear spots on an opaque white background the latter being generated by precipitation of SDS with zinc ions This negative staining procedure is sensitive fast and completely reversible upon removal of zinc with a chelating agent Materials Polyacrylamide gel containing protein s of interest See UNIT 6 1 GelCode E Zinc Reversible Stain Kit Pierce containing E Zinc Stain E Zinc Developer E Zinc Eraser Distilled water Plastic container with lid pipet tip containers are appropriate for staining mini gels Platform shaker optional NOTE All steps should be performed at room temperature Stain the gel 1 Remove polyacrylamide gel from electrophoresis assembly and place it in a plastic container with lid containing 10 gel vol E Zinc Stain solution Incubate 10 min with gentle agitation The use of a platform shaker is recommended Current Protocols in Cell Biology 3 Remove the E Zinc Stain solution from the container and replace it with 10 gel vol E Zinc Developer solution Do not reuse the stain solution 4 Incubate 1 to 2 min with gentle agitation Proceed to the next step as soon as the staining is judged to be optimal 5 Remove the E Zinc Developer solution from the container and replace it with 10 gel vol distilled water Do not reuse the developer solution Wash the gel 6 Incubate 1 min with gentle agitation 7 Change the water in the container 8 V
298. ncentra tion of primary antibody should decrease back ground and improve specificity Fig 6 2 3 Due to the nature of light and the method of detection certain precautions are warranted when using luminescent visualization e g Harper and Murphy 1991 Very strong signals can overshadow nearby weaker signals on the membrane Because light will pipe through the membrane and the surrounding plastic wrap overexposure will produce a broad diffuse im Current Protocols in Cell Biology age on the film The signal can also saturate the film exposing the film to a point whereby increased exposure will not cause a linear in crease in the density of the image on the film With the alkaline phosphatase substrate AMPPD nitrocellulose PVDF and nylon membranes require 2 4 and 8 to 12 hr respec tively to reach maximum light emission In addition PVDF is reported to give a stronger signal than nitrocellulose Tropix Western Light instructions Positively charged nylon requires special blocking procedures to mini mize background Gillespie and Hudspeth 1991 These procedures include using a block ing and primary antibody solution containing 6 casein 1 polyvinylpyrrolidone 40 PVP 40 3 mM NaN3 10 mM EDTA and PBS pH 6 8 Prior to use the casein must be heated to 65 C to reduce alkaline phosphatase activity in the casein itself In addition maximum sensi tivity has been observed when free biotin or biotinylated proteins
299. ncentrations e g 5 to 20 and 10 to 20 acrylamide can be cast and stored giving much more flexibility to optimize separations Current Protocols in Cell Biology SUPPORT PROTOCOL 2 Electrophoresis and Immunoblotting ee 6 1 21 Supplement 37 One Dimensional SDS PAGE 6 1 22 Supplement 37 Additional Materials also see Alternate Protocol 5 Plug solution see recipe Light and heavy acrylamide gel solutions for multiple gradient gels Table 6 1 11 TEMED H2O saturated isobutyl alcohol Multiple gel caster Bio Rad Hoefer Peristaltic pump 25 ml min 500 or 1000 ml gradient maker Bio Rad Hoefer Tygon tubing Set up system and pour separating gel 1 Assemble the multiple caster as in casting multiple single concentration gels see Support Protocol 1 steps 1 to 3 making sure to remove the triangular space filler plugs in the bottom of the caster The plug is used only when casting single concentration gels Table 6 1 11 Light and Heavy Acrylamide Gel Solutions for Casting Multiple Gradient Gels Acrylamide concentration in light gel solution Stock solution 5 6 7 8 9 10 11 12 13 14 30 acrylamide 0 8 28 33 39 44 50 55 61 66 Nn 77 bisacrylamide 4x Tris Cl SDS 41 41 41 41 41 A 41 A 41 41 pH 8 8 H20 96 91 85 80 74 69 63 58 52 47 10 ammonium 0 55 0 55 0 55 0 55 0 55 0 55 0 55 0 55 0 55 0 55 persulfate TEMED 0 054 0 054 0 054 0 054 0 054 0 054 0 054 0 054 0 054
300. nd whether a soluble ampholyte system or an Immobiline gel will be used The major consideration affecting appropri ate gel size is the degree of resolution needed In general the smallest gel format shouldbe selected that will provide the needed degree of resolution because smaller gels are easier less expensive and faster to run Therefore quick screening of samples or analysis of relatively simple samples can easily be accomplished with microgels or minigels In contrast if detailed qualitative or quantitative comparisons of cell or tissue extracts are planned standard size or large gels are indi cated Similarly 3 mm or larger first dimension tube gels followed by 1 5 mm second dimension gels in the standard or large format are indicated if the two dimensional gels will be used for prepa rative isolation of a protein for applications such as raising antibodies or conducting structural analysis Immobilized pH gradient gels should very seriously be considered as an alternative to soluble ampholyte gels for most separations ow ing to their stable and reproducible pH profiles The IPG gels are particularly appropriate when a narrow pH range is required Immobilized pH gradient gels are typically run at 2500 to 3500 V and typically require a focusing time of 16 to 18 hr Use of high voltage 8 000 to 10 000 V integrated power supplies cooling units can shorten focusing times to lt 4 hr Optimal focusing conditions may be experiment
301. ndard conditions UNIT 6 2 Current Protocols in Cell Biology NATIVE TRANSFER OF THE PROTEINS FROM THE FIRST DIMENSION BN PAGE GEL TO A MEMBRANE FOR IMMUNOBLOTTING Proteins are separated by BN PAGE under native conditions Transfer to a membrane and visualization by immunoblotting can also be performed under native conditions If a cell lysate is analyzed a very specific antibody that does not cross react with other proteins is necessary The antibodies that are used for immunodetection after SDS PAGE might not be suitable since they recognize unfolded proteins Antibodies that recognize folded proteins and protein complexes as used for immunoisolation e g immunoprecipitation flow cytometry or immunofluorescence methods should be tested for the application in this protocol In this protocol the proteins multiprotein complexes are transferred to a membrane in their native form The following immunoblotting procedure is similar to the standard protocol used after SDS PAGE UNIT 6 2 Materials First dimension BN PAGE gel Basic Protocol 1 Native BN transfer buffer see recipe Destaining solution see recipe PVDF membrane see UNIT 6 2 Additional reagents and equipment for immunoblotting UNIT 6 2 1 Briefly rinse the first dimension BN gel in native BN transfer buffer 2 Transfer the native proteins to a PVDF membrane by semi dry or wet blotting UNIT 6 2 Use the native BN transfer buffer since it does not contain SD
302. nge Metal or plastic scoop Dry ice pellets Wash tubes and prepare the gel mixture 1 Remove the glass tubes from a chromic acid filled container Extensively wash the tubes with water using high purity water for the last wash Dry the tubes at least 1 hr in an oven at 110 C and store them at room temperature covered with aluminum foil To prevent gels from sticking to the glass tubes gel tubes have to be very clean Satisfactory results are obtained by storing the tubes in chromic acid between uses and washing them shortly before use Because drying the tubes requires at least 1 hr cleaning steps should be performed the day before gels will be cast CAUTION Chromic acid is highly corrosive follow supplier s precautions carefully 2 Prepare the gel solution by mixing 16 9 g urea 4 0 ml of 30 acrylamide 0 8 bisacrylamide 3 0 ml of 20 w v Triton X 100 7 5 ml water 3 0 ml ampholytes Briefly warm the mixture in a 37 C water bath to solubilize urea if needed To minimize decomposition of urea never warm any solutions containing urea above 37 C use ultrapure urea and prepare solutions immediately before use Choice of ampholyte composition is one of the key factors determining the quality of isoelectric focusing separations Substantial differences in performance resolution and shape of the pH gradient formed may be observed with different combinations of ampho lytes and with ampholytes from different suppliers
303. node buffer in the lower buffer chamber The cathode buffer contains the tricine 4 Connect the power supply to the cell and run 1 hr at 30 V constant voltage followed by 4 to 5 hr at 150 V constant voltage Use a heat exchanger to keep the electrophoresis chamber at room temperature 5 After the tracking dye has reached the bottom of the separating gel disconnect the power supply Refer to Safety Considerations under Electricity and Electrophoresis Coomassie blue G 250 is used as a tracking dye instead of bromphenol blue because it moves ahead of the smallest peptides 6 Disassemble the gel see Basic Protocol 1 steps 23 to 26 Stain proteins in the gel for 1 to 2 hr in Coomassie blue G 250 staining solution Follow by destaining with 10 acetic acid changing the solution every 30 min until background is clear 3 to 5 changes For higher sensitivity use silver staining as a recommended alternative Prolonged staining and destaining will result in the loss of resolution of the smaller proteins lt 10 kDa Proteins diffuse within the gel and out of the gel resulting in a loss of staining intensity and resolution NONUREA PEPTIDE SEPARATIONS WITH TRIS BUFFERS A simple modification of the traditional Laemmli buffer system presented in Basic Protocol 1 in which the increased concentration of buffers provides better separation between the stacked peptides and the SDS micelles permits reasonable separation of peptides as sma
304. nsifying screen EES film sample film cassette Figure 6 3 1 Autoradiography setup intensifying screen film and sample in film cassette 3 Expose the film for the desired length of time and at the appropriate temperature Time of exposure will depend on the strength of the radioactivity in the sample and in most cases will have to be determined empirically by making multiple exposures for different lengths of time To help estimate exposure time a Geiger counter can often be used to detect the relative amount of radioactivity in the sample With experience this can help alleviate the trial and error often associated with obtaining the optimum exposure Time of exposure and use of internal controls are particularly important if quantitative comparisons between experiments are desired 4 After exposure return cassette to the darkroom and remove film for developing If the film was exposed at 70 C allow the cassette to come to room temperature before developing This will avoid static discharge which can cause black dots or stripes on the autoradiogram Automated film developers are also available and can be used to develop the film 5 Immerse the film for 5 min in 18 to 20 C developer then wash min in running water at room temperature Shorter periods of time in developer will yield a lighter image The amount of time in developer therefore can be used to roughly control intensity of th
305. nsylvania Current Protocols in Cell Biology One Dimensional Electrophoresis Using Nondenaturing Conditions Nondenaturing or native electrophoresis i e electrophoresis in the absence of dena turants such as detergents and urea is an often overlooked technique for determining the native size subunit structure and optimal separation of a protein Because mobility depends on the size shape and intrinsic charge of the protein nondenaturing electropho resis provides a set of separation parameters distinctly different from mainly size depend ent denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS PAGE UNITS 6 1 and charge dependent isoelectric focusing IEF unir 6 4 Two protocols are presented below Continuous PAGE see Basic Protocol is highly flexible permitting cationic and anionic electrophoresis over a full range of pH The discontinuous procedure see Alternate Protocol is limited to proteins negatively charged at neutral pH but provides high resolution for accurate size calibration CONTINUOUS ELECTROPHORESIS IN NONDENATURING POLYACRYLAMIDE GELS Separation of proteins by nondenaturing electrophoresis requires the same type of equipment used for denaturing slab gels unr 6 1 and is adaptable to a range of gel sizes e g from 7 3 x 8 3 cm minigels to 14 x 16 cm full size gels and matrix types e g single concentration and gradient gels This protocol outlines straightforward proce dures
306. nt This type of electrophoresis is thus referred to as SDS polyacy lamide gel electrophoresis SDS PAGE Denaturation of the proteins prior to electrophoresis allows for enhanced resolution and discrimination of proteins on the basis of molecular size rather than charge or shape Utilization of a discontinuous system i e the apposition of stacking and separating gels results in concentration of dilute samples and enhanced band sharpness unr 6 1 presents an overview of electricity and electrophoresis followed by detailed protocols for SDS PAGE using either Laemmli s buffers and gel system or modifi cations of this system i e use of Tris tricine buffers higher concentrations of buffers gradient gels single concentration gels and minigels The unit also explains how to calculate the apparent molecular weights of proteins from SDS PAGE data The next unit in the chapter var 6 2 describes protocols for immunoblotting also referred to as western blotting In this technique proteins separated by any of the electrophoretic techniques described in unr 6 1 are electrophoretically transferred electroblotted onto a membrane The membrane which thus becomes a replica of the polyacrylamide gel is subsequently probed with antibodies to specific proteins The primary antibodies can be revealed by an additional incubation with I labeled secondary antibodies or protein A followed by autoradiography unrr 6 3 In recent years
307. ntire stacking gel or use a two dimen sional comb Most two dimensional gel combs have a separate small well for a standard or reference sample The use of beveled plates see Basic Protocol 3 steps 1 to 3 is not essential but is still preferred because it will facilitate loading of the first dimension gel In this procedure the first dimension gel will fit between the glass plates if 1 2 mm tubes are used for the first dimension and 1 5 mm gels are used for the second dimension 8 Load the first dimension gel onto the second dimension gel Remove any air bubbles trapped between the gels If the first dimension gel does not remain securely in place it can be embedded using 1 5 w agarose in reducing buffer 9 Carefully pour electrophoresis buffer into the upper electrophoresis chamber and electrophorese using voltages and times appropriate for the gel type selected Parameters for electrophoresis are given in UNIT 6 1 USING TWO DIMENSIONAL PROTEIN DATABASES Computerized image acquisition and manipulation constitute the only practical method for systematic qualitative and quantitative evaluation of complex protein patterns from different samples that are to be compared by high resolution two dimensional gel analy sis Examples of experimental applications include comparisons of tumor cells or tissues with appropriate normal controls and comparisons of a single cell line under different experimental conditions There are curre
308. ntly a number of commercially available image acquisition computer systems specifically designed for comparing two dimensional gels and storing associated information in a database The systems include both hardware and the necessary software for comparing different gels and producing databases containing the two dimensional protein patterns with options for annotating specific spots and producing quantitative comparisons among large numbers of different samples With most systems images can be acquired from either stained gels or autoradiographs The equipment used to obtain two dimensional gel images includes laser scanners video cameras and phosphoimagers After image acquisition software running on a microcomputer or workstation is used to refine the image detect spots and match spots between different gels It is essential that very high quality reproducible gels be used for computerized compari sons The greatest dynamic range in protein abundance for a single two dimensional gel can be obtained using autoradiography or phosphoimaging Un7T6 3 With these methods up to several thousand spots can be compared and tracked A representative reference gel or a composite image can be stored and used as a reference for future experiments Information related to each spot on the two dimensional pattern including the quantity of protein in the indicated spot on different gels used in the comparison can be archived and updated Other known inform
309. ntributed by Dennis M Krizek and Margaret E Rick Current Protocols in Cell Biology 2002 6 7 1 6 7 13 Copyright 2002 by John Wiley amp Sons Inc UNIT 6 7 BASIC PROTOCOL Electrophoresis and Immunoblotting 6 7 1 Supplement 15 Agarose Gel Electrophoresis of Proteins 6 7 2 Supplement 15 apparatus UNIT 6 2 and the proteins are transferred overnight onto an immobilization matrix PVDF membrane The membrane is blocked and probed with a polyclonal primary antibody specific for the antigen of interest and after washing a conjugated secondary antibody is introduced After incubation the membrane is washed again and the protein bands of interest are illuminated with a chemiluminescent detection system Materials SeaKem HGT P agarose Bio Whittaker or equivalent 1x electrophoresis buffer 4 C see recipe Protein samples 2x sample buffer see recipe 0 25x transfer buffer without methanol see recipe Blocking buffer UNIT 6 2 containing 5 w v nonfat dry milk fresh Antibodies Primary rabbit anti vWF Dako Secondary donkey horseradish peroxidase linked anti rabbit Ig Amersham Pharmacia ECL Western Blotting Analysis system Amersham Pharmacia Aluminum foil Boiling waterbath optional Horizon 20 25 horizontal electrophoresis apparatus Life Technologies or equivalent Teflon comb e g 1 x 9 mm 20 well Pipet with fine tip or equivalent 0 45 um Immobilon P polyvinylidene fluo
310. o digital converter ADC The method of image assembly depends on the light source and detector geometry One method is to capture the image all at once using a two dimensionally arrayed CCD detector similar to the detectors found in digital and video cameras Typically a camera type sensor is paired with a light source that evenly illumi nates the sample This same sensor is often used with fluorescent and chemiluminescent detec tion methods as its ability to detect light con Current Protocols in Cell Biology tinuously over the entire sample reduces image capture times Another method of image assem bly is to capture the image a line at a time This typically involves a linearly arrayed CCD scan ning slowly across the sample in conjunction with the detection beam of light The data from each line is then compiled into a composite image Spatial resolution in this method can be significantly better on large format samples than the resolution of a camera based system This method is also advantageous when OD based detection is used since the more focused light beam is usually of higher intensity and can penetrate denser material A third method of image assembly is to use a point light source and single element detector on each point on a sample The image is then compiled from each point sampled This method is slower than the others but can offer extremely high resolution and sensitivity A fourth commonly encoun tered method is
311. ocess can enhance visualization of specific features but is best left to adjustments in look up tables LUTs in later analysis steps rather than during image capture since there is a risk of data loss during postacquisition image processing LUTs are indexed palettes or tables where each index value corresponds to color or gray scale intensity values present in an image Many image analysis programs alter LUTs instead of image values directly since it both is faster and does not change the original image data Once all the capture parameters are opti mized the image capture process is initiated This might take less than a second for images captured with camera based detectors and up to hours for scanning single point detectors When the image has been captured it should be carefully examined for content It should fully capture the area of interest and the parame ters should have been set so that all necessary information is detectable Furthermore it should be in a form that will allow for easy analysis Extra time spent optimizing the cap ture parameters will often result in a reduction in total image analysis time and in an increase in data quality When the best possible image has been captured it often contains information outside the area of interest While this is un likely to cause problems with later analysis it is often advantageous to crop the image so that the only portion that is saved contains the area of interest T
312. ocols in Cell Biology BASIC PROTOCOL 2 Electrophoresis and Immunoblotting 6 6 3 Supplement 6 Staining Proteins in Gels 6 6 4 Supplement 6 Plastic container with lid pipet tip containers are appropriate for staining mini gels Clean plastic containers at least six lids of pipet tip containers are appropriate for staining mini gels Aluminum foil Platform shaker optional CAUTION Glacial acetic acid and methanol are volatile and toxic solutions containing these solvents should be prepared in a chemical fume hood and handled with care NOTE All solutions should be prepared in deionized water Milli Q or HPLC grade At all times wear gloves that have been rinsed extensively with distilled water Handle the gel by using clean forceps having blunt tips or by touching the corners with clean powder free gloves All steps should be performed at room temperature Fix the gel 1 Remove polyacrylamide gel from electrophoresis assembly and place it in a plastic container with lid containing a large excess 10 gel vol of fixative solution Steps 1 and 2 are for unfixed gels only If the polyacrylamide gel was previously fixed and or stained with Coomassie blue Basic Protocol 1 or Alternative Protocol 1 proceed directly to step 3 2 Incubate for 230 min with gentle agitation The use of a platform shaker is recommended Pretreat the gel 3 Transfer the gel to a clean plastic container having 10 gel
313. ocols in Cell Biology Additional Materials also see Basic Protocol 3 Luminescent substrate buffer 50 mM Tris Cl pH 7 5 HARPO APPENDIX 24 or dioxetane phosphate substrate buffer alkaline phosphatase see recipe Nitro Block solution AP reactions only 5 v v Nitro Block Tropix in dioxetane phosphate substrate buffer prepared just before use Luminescent visualization solution Table 6 2 1 Clear plastic wrap Additional reagents and equipment for autoradiography UNIT 6 3 NOTE See Troubleshooting section for suggestions concerning optimization of this protocol particularly when employing AP based systems 1 Equilibrate membrane in two 15 min washes with 50 ml substrate buffer For blots of whole gels use 50 ml substrate buffer for strips use 5 to 10 ml strip For AP reactions using nitrocellulose or PVDF membranes Incubate 5 min in Nitro Block solution followed by 5 min in substrate buffer volumes as in step 1 Nitro Block enhances light output from the dioxetane substrate in reactions using AMPPD CSPD or Lumigen PPD concentrate It is required for nitrocellulose and recommended for PVDF membranes It is not needed for Lumi Phos 530 AP reactions on nylon membranes or HRPO based reactions on any type of membrane Lumi Phos 530 is not recommended for nitrocellulose membranes Transfer membrane to visualization solution Soak 30 sec HRPO reactions to 5 min AP reactions volumes as in step 1 Alternat
314. odium phosphate 0 4 wiv SDS Mix 46 8 g NaH2PO 4 H20 231 6 g NagHPO 7H20 and 12 g SDS in 3 liters H20 Store at 4 C for up to 3 months The recipes produce 15 ml of separating gel which is adequate for one gel of dimensions 0 75 mm x 14 cm x 14 cm The recipes are based on the original continuous phosphate buffer system of Weber et al 1972 Pall reagents and solutions used in the protocol must be prepared with Milli Q purified water or equivalent Volumes are in milliliters The desired percentage of acrylamide in the separating gel depends on the molecular size of the protein being separated See Basic Protocol 1 annotation to step 3 One Dimensional 4Best to prepare fresh Failure to form a firm gel usually indicates a problem with the ammonium persulfate TEMED SDS PAGE or both 6 1 14 Supplement 37 Current Protocols in Cell Biology 1 Prepare and pour a single separating gel see Basic Protocol 1 steps 1 to 4 except use the recipe in Table 6 1 8 and fill the gel sandwich to the top Omit the stacking gel Insert the comb see Basic Protocol 1 step 10 and allow the gel to polymerize 30 to 60 min at room temperature 2 Mix the protein sample 1 1 with 2x phosphate SDS sample buffer and heat to 100 C for 2 min For large sample volumes or samples suspended in high ionic strength buffers gt 50 mM dialyze against 1 x sample buffer prior to electrophoresis Note that the precautions about proteases see Basic Protocol 1 step
315. of 2 cm below the top of the beveled plate to accommodate the stacking gel 3 After the separating gel has polymerized a sharp interface between the polymerized gel and the water overlay will reappear remove the overlay rinse the gel surface with water and pour the stacking gel The stacking gel solution should reach to the top of the bevel Immediately overlay the stacking gel solution with a minimum amount of water which will adhere owing to the surface tension see Fig 6 4 2A A water overlay of the stacking gel provides a smooth surface and better contact between the IEF gel and second dimension gel A small volume of water has to be used to avoid lowering the upper edge of the stacking gel below the edge of the beveled plate The stacking gel height must be between 1 5 and 2 cm The solution is filled to the top of the bevel so that after the slight shrinkage that occurs during polymerization the top of the polymerized gel will be near the bottom of the bevel see Fig 6 4 2B Load the isoelectric focusing gels onto the second dimension gels 4 Assemble second dimension gels in an electrophoresis chamber Do not pour elec trophoresis buffer into the upper chamber 5 Melt 2 w v agarose in a boiling water bath and add an equal volume of equilibra tion buffer for use in step 11 Keep agarose equilibration buffer in the boiling water bath until step 11 is completed Current Protocols in Cell Biology Electrophoresis and Imm
316. of equilibration buffer For solution 2 Add 0 45 g iodoacetamide and a few grains bromphenol blue per 10 ml of equilibration buffer Make fresh immediately before use Final concentrations are 50 mM Tris Cl pH 6 8 6 M urea 30 glycerol and 1 SDS in a final volume of 200 ml Current Protocols in Cell Biology Electrophoresis and Immunoblotting 6 4 27 Supplement 4 EDTA 2 w 2 g Na EDTA H O to 100 ml Adjust to pH 7 0 with NaOH Store at room temperature stable several months Titrate while dissolving EDTA is difficult to dissolve without addition of NaOH even when the disodium salt is used Equilibration buffer 3 g SDS 7 4 ml 2 w v EDTA pH 7 0 see recipe 10 ml glycerol 2 ml 1 0 M Tris Cl pH 8 65 APPENDIX 24 0 3 ml bromphenol blue saturated solution in H O H O to 100 ml Store at room temperature stable for several weeks Final concentrations are 3 w v SDS 0 4 mM EDTA 10 v v glycerol and 20 mM Tris Cl pH 8 65 Leupeptin 2 mg ml 20 mg leupeptin 10 ml water Divide into convenient volumes Store at 20 C stable at least 1 year Lysis buffer 2 59 g urea ultrapure 1 6 ml H O 0 25 ml 2 mercaptoethanol 0 3 ml ampholytes 1 0 ml 20 w v Triton X 100 solution see recipe Prepare immediately before use Use same ampholytes as for the IEF gel formulation To dissolve urea warm the mixture in a 30 C water bath if necessary Orthophosphoric acid H POy 0 1 M 13 7 ml 85 phosphoric acid W
317. of protein glycosylation is readily accomplished by polyacrylamide gel electrophoresis Koch and Smith 1990 Packer et al 1997 Taverna et al 1998 Koketsu and Linhardt 2000 However relatively few high sensitivity methods have been developed to reliably detect oligosaccharide residues cova lently attached to proteins for visualization in polyacrylamide gels or on electroblot mem branes Packer et al 1997 Packer et al 1999 Koketsu and Lindhardt 2000 Perhaps the most common procedure to visualize glycopro teins reported in the literature entails detection by periodic acid Schiff PAS staining using the colorimetric acid fuchsin dye A major limita Current Protocols in Cell Biology tion of this method is that detection sensitivity is poorer than Coomassie Blue staining ren dering the technique obsolete for modern high sensitivity proteomics investigations Other methods in use include PAS labeling with digoxigenin hydrazide followed by immunode tection with anti digoxigenin antibody conju gated to alkaline phosphatase or PAS labeling with biotin hydrazide followed by detection with horseradish peroxidase or alkaline phos phatase conjugated to streptavidin Packer et al 1995 Lectins are commonly employed to detect certain structural subclasses of glycopro teins by methods similar to those employed in immunoblotting Koketsu and Lindhardt 2000 All of these methods require that pro Electrophoresis an
318. of the gel NONEQUILIBRIUM ISOELECTRIC FOCUSING OF VERY ACIDIC PROTEINS Basic Protocol 1 is sufficient for separating proteins with isoelectric points greater than 3 5 to 4 0 For very acidic proteins however a nonequilibrium system is needed The major features of this method are utilization of a shorter focusing time without reaching equilibrium a modified ampholyte mixture and different electrode solutions Additional Materials also see Basic Protocol 1 10 w v ammonium persulfate prepare immediately before use Concentrated sulfuric acid used in lower chamber electrode solution Ampholytes pH 2 11 used in upper chamber electrode solution To analyze very acidic proteins follow Basic Protocol 1 with these exceptions in the indicated steps 2 When preparing the gel solution use the following mixture of ampholytes 2 4 ml ampholytes pH 2 5 4 and 0 6 ml ampholytes pH 2 11 4 Following the procedure for casting gels by pouring add 100 ul of 10 ammonium persulfate solution swirl add 42 5 ul TEMED and swirl again Gel mixtures containing entirely or predominantly very acidic or very basic ampholytes are generally difficult to polymerize Use of an increased ammonium persulfate concentra tion and adherence to the proper order of adding the reagents should ensure polymeriza tion 8 Prepare the bottom chamber electrode solution by adding 4 5 ml concentrated sulfuric acid to 3 liters water Degas at least 5 min O
319. of the mitochondrial outer membrane Nat Struct Biol 8 361 370 Poetsch A Neff D Seelert H Schagger H and Dencher N A 2000 Dye removal catalytic activity and 2D crystallization of chloroplast H ATP synthase purified by blue native elec trophoresis Biochim Biophys Acta 1466 339 349 Rexroth S Meyer zu Tittingdorf J M Krause F Dencher N A and Seelert H 2003 Thylakoid membrane at altered metabolic state Challeng ing the forgotten realms of the proteome Elec trophoresis 24 2814 2823 Schamel W W Arechaga I Risueno R M van Santen H M Cabezas P Risco C Valpuesta J M and Alarcon B 2005 Coexistence of mul tivalent and monovalent TCRs explains high sensitivity and wide range of response J Exp Med 202 493 503 Current Protocols in Cell Biology Schagger H 1995 Quantification of oxidative phosphorylation enzymes after blue native elec trophoresis and two dimensional resolution Normal complex I protein amounts in Parkin son s disease conflict with reduced catalytic ac tivities Electrophoresis 16 763 770 Schagger H and von Jagow G 1991 Blue na tive electrophoresis for isolation of membrane protein complexes in enzymatically active form Anal Biochem 199 223 231 Schagger H Cramer W A and von Jagow G 1994 Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of mem brane protein
320. ographic images Johnston et al 1990 They can detect radioiso topes such as P 1 4C S and H There are several advantages of phosphor imaging over film 1 linear dynamic ranges are 5 orders of magnitude compared to 1 5 orders of magnitude for film Fig 6 3 2 2 exposure times are 10 to 250 times faster than with film 3 quantification is much easier and faster and most imagers come with software to directly analyze data 4 fluorography and gel drying are often unnecessary because of the sensitivity of phosphor imaging and 5 phosphor screens can be reused indefinitely if handled carefully Phosphor imaging screens are composed of crystals of BaFBr Eu When the screen is exposed to ionizing radiation such as o B or y radiation or wavelengths of light shorter than 380 nm the electrons from Eu are excited and then trapped in an F center of the BaFBr complex this results in the oxidation of Eu to Eu which forms the latent image on the screen After exposure the latent image is released by scanning the screen with a laser 633 nm During scanning Eu reverts back to Eu releasing a photon at 390 nm The luminescence can then be collected and measured in relation to the position of the scanning laser beam The result is a representation of the latent image on the storage phosphor imaging plates The image can then be viewed on a video monitor and analyzed with the aid of appropriate software Som
321. ohn Wiley amp Sons Inc UNIT 6 8 Electrophoresis and Immunoblotting 6 8 1 Supplement 16 BASIC PROTOCOL 1 Fluorescence Detection of Glycoproteins in Gels and on Electroblots 6 8 2 Supplement 16 FLUORESCENT DETECTION OF GLYCOPROTEINS IN POLYACRYLAMIDE GELS Pro Q Emerald 300 Glycoprotein Gel Stain Kit provides a robust method for differentially staining glycosylated and non glycosylated proteins in the same gel The technique combines the green fluorescent Pro Q Emerald 300 glycoprotein stain with the orange red fluorescent SYPRO Ruby total protein gel stain Pro Q Emerald 300 glycoprotein stain reacts with periodate oxidized carbohydrate groups creating a bright green fluorescent signal on glycoproteins Using this stain allows detection of lt 1 ng glycoprotein band depending upon the nature and the degree of glycosylation making it 100 fold more sensitive than the standard periodic acid Schiff base method using acidic fuchsin dye rosaniline The green fluorescent signal from the Pro Q Emerald 300 stain can be visualized using a standard 300 nm UV UV B illumination source The Pro Q Emerald 488 Glycoprotein Gel Stain Kit is quite similar to the Pro Q Emerald 300 Glycoprotein Gel Stain Kit but it is optimized for use with gel scanners equipped with 470 to 488 nm lasers The Pro Q Emerald dye staining method is more reliable than mobility shift assays using glycosidases since even glycoproteins that
322. oligan B M Dunn H L Ploegh D W Speicher and P T Wingfield eds pp 10 5 1 10 5 12 John Wiley amp Sons New York Fernandez Patron C Calero M Collazo P R Garc a J R Madrazo J Musacchio A Sori Electrophoresis and Immunoblotting 6 6 13 Supplement 6 Staining Proteins in Gels 6 6 14 Supplement 6 ano F Estrada R Frank R Castellanos Serra L R and Mendez E 1995a Protein reverse staining High efficiency microanalysis of un modified proteins detected on electrophoresis gels Anal Biochem 224 203 211 Fernandez Patron C Hardy E Sosa A Seoane J and Castellanos Serra L R 1995b Double staining of Coomassie blue stained polyacry lamide gels by imidazole sodium dodecyl sulfate zinc reverse staining sensitive detection of Coomassie blue undetected proteins Anal Bio chem 224 263 269 Garfin D E 1990 One dimensional gel electropho resis Methods Enzymol 182 425 441 Hitchman M L and Ekstrom B 1994 An investi gation of the factors controlling the staining and destaining of electrophoresis gels Electrophore sis 15 200 208 Na D S Hong H Y Yoo G S and Choi J K 1994 Evans blue staining method for detection of proteins on polyacrylamide gels with rho damine B Anal Lett 27 1265 1275 Neuhoff V Stamm R and Eibl H 1985 Clear background and highly sensitive protein staining with Coomassie blue dyes in polyacrylamid
323. ology Typical search parameters include ones for de tection sensitivity smoothing minimum inter band gaps and minimum or maximum band peak size Smoothing reduces the number of bands detected due to noise in the image A minimum interband gap is often used to avoid detection of false secondary bands on the shoul ders of primary bands Limitations on peak sizes especially for within lane comparison to the largest band s peak can be a useful way to allow sensitive detection of bands in under loaded lanes without detecting false bands in overloaded lanes Band edges are often detected in addition to band peaks in order to further define bands or to quantitate band amounts This can be accom plished by using local minima derivatives of the lane profile or fixed parameters such as image distances or a percentage of band peak height The band edges can be applied as edges perpendicular to the long axis of the lane or as a contour of equal intensity circling the band The perpendicular method is advantageous for bands with uneven distribution of material across the face of the band while the latter method is better for smiling or misshapen bands Background subtraction With nearly all electrophoresis procedures the most informative images have a low level of signal intensity at each pixel that does not result from protein or nucleic acid Instead this background intensity is attributable to the gel medium the visualizat
324. olution protects samples from direct contact with the strong base used as an upper electrode solution Dilution of the lysis buffer with water is necessary to decrease the density so the overlay does not mix with the sample A 3 mm i d x 16 cm long IEF gel has a total protein capacity of 500 ug for whole cell extracts and other complex protein mixtures The maximum capacity for any single protein spot is 0 5 to 5 ug depending on its solubility near its isoelectric point and the separation distance from any near neighbors Preparation of relatively pure protein samples for Current Protocols in Cell Biology Electrophoresis and Immunoblotting 6 4 5 Supplement 4 Two Dimensional Gel Electrophoresis 6 4 6 Supplement 4 23 24 isoelectric focusing is generally straightforward The sample usually may be prepared in one of the following ways dialyze into any compatible low ionic strength buffer lyophilize in a volatile or compatible low ionic strength buffer and dissolve in lysis buffer or precipitate the protein using trichloroacetic acid TCA and redissolve in lysis buffer For preparing extracts from cultured cells and from tissue samples see Support Protocol 4 and Support Protocol 5 respectively The minimum sample concentration of protein or radioactivity has to be sufficient for the desired detection method For complex protein mixtures such as tissue or cell extracts a 500 ug total load is recommended fo
325. ommonly encountered in ordinary use First the film should be preexposed to a hypersensitizing flash of light which provides several photons per silver bromide crystal and stably activates them without providing enough exposure to cause them to become developed This allows a linear relationship to be drawn between the film image and the amount of radioactivity in the sample Second film exposure should be conducted at low tempera tures 70 C to slow the reversal of activated silver bromide crystals to their stable form Laskey and Mills 1975 Film can be hypersensitized by exposure to a flash of light lt 1 msec provided by a photographic flash unit or a stroboscope before being placed onto the radioactive sample for exposure of the autoradiogram Laskey and Mills 1975 1977 As the optimal light intensity required for preexposure varies with the type of film and the flash unit being used the ideal exposure is best determined empirically as described below Materials Stroboscope or flash unit e g Auto 22 Electronic Flash from Vivitar or Sensitize Pre Flash from Amersham Pharmacia Biotech Neutral density filter Kodak Orange filter Wratten 22 Kodak X ray film Spectrophotometer 1 Cover the stroboscope or flash unit with the neutral density and orange filter This serves to decrease the intensity of emitted light particularly the blue wavelengths to which X ray films are most sensitive Filters are not required for the Amers
326. on gels 7 Using forceps remove the IPG gels from the electrophoresis tray after isoelectric focusing is complete or from the 80 C freezer see Basic Protocol 2 step 19 and place each strip in a separate petri dish with the support film side of the strip facing the petri dish wall Add 15 ml of DryStrip equilibration buffer 1 Cover and place on a platform shaker for 10 min Strips may be run in the second dimension immediately after isofocusing or after storage at 80 C If the strips have been stored at 80 C remove them from the freezer then place in petri dish as stated and continue with the equilibration procedure 8 Discard equilibration buffer 1 and add 15 ml of equilibration buffer 2 Cover and place on a platform shaker for 10 min 9 Dampen a piece of filter paper and place on a glass plate Remove the DryStrips from equilibration buffer 2 Place each strip on its edge on the filter paper to remove any excess buffer Strips should not be left in this position for gt 10 min or spot sharpness may be affected Current Protocols in Cell Biology Electrophoresis and Immunoblotting 6 4 23 Supplement 4 SUPPORT PROTOCOL 6 Two Dimensional Gel Electrophoresis 6 4 24 Supplement 4 10 Add a small amount of SDS electrode buffer along the glass plate above the second dimension gel Place the DryStrip gel in the well with the gel facing out and the basic side to the left Push the DryStrip down
327. ons Determination of the migration of proteins on parallel gels made up of different concentrations of acrylamide and bisacrylamide allows calculation of their molecular weights using Ferguson plots Proteins separated by electrophoresis can be visualized by direct staining of the gels Four procedures for staining proteins in gels based on different principles are presented in UNIT 6 6 These procedures involve staining with Coomassie blue silver SYPRO ruby or zinc ions The unit describes the basic protocols and provides guidelines for the selection of a specific protocol The separation of proteins by polyacrylamide gel electrophoresis is limited to proteins with molecular weights less than 300 000 The electrophoretic separation of larger proteins or multiprotein complexes requires the use of other matrix materials A suitable material is agarose which is most commonly used for the separation of DNA UNIT 6 7 presents protocols for the electrophoretic separation of proteins on agarose gels In Current Protocols in Cell Biology addition to permitting the separation of very large proteins these protocols allow the analysis of multimerization or aggregation Although these processes can also be analyzed by sedimentation on sucrose gradients UNIT 6 3 or gel filtration UNIT 6 4 agarose gel electrophoresis is more convenient for analysis of large numbers of samples LITERATURE CITED Laemmli U K 1970 Cleavage of structural proteins dur
328. or of each well It is helpful to construct a template to place under the gel to facilitate punching the wells in an evenly spaced straight line Also it is necessary to work quickly because these gels are very thin and dry out rapidly Place a glass plate across the bridge on the electrophoresis chamber and place the gel attached to the GelBond on top of the plate Affix the presoaked wicks step 13 on each side of the gel so that there is continuity between the agarose running buffer in the electrode chambers and the gel Current Protocols in Cell Biology 17 Load 8 ul of sample into each well Place the cover over the electrophoresis apparatus and connect the power supply 18 Electrophorese at 25 V for 30 min at room temperature to allow the samples to enter the gel matrix Increase the power supply to 50 V and continue electrophoresis until the dye marker has migrated 8 to 10 cm from the wells 3 to 5 hr Fix gel 21 Add 200 ml fixing buffer to a container appropriate to the size of the gel Carefully remove the paper wicks and place the gel in fixing buffer 1 hr without agitation The gel can also fix overnight in fixing buffer for a convenient stopping point 22 Wash the gel 1 hr with 200 to 300 ml BSB with gentle mixing on a horizontal rotary mixer A prerinse to 2 min before the wash with 100 ml BSB is recommended 23 Prepare 250 ml fresh in gel blocking buffer Block gel 1 hr with gentle mixing 24 Prerinse gel with
329. ositive anode However some proteins may be positively charged An additional membrane placed on the cathode side of the gel will bind these proteins 6 Prepare transfer membrane Cut membrane to same size as gel plus 1 to 2 mm on each edge Place into distilled water slowly with one edge at a 45 angle Equilibrate 10 to 15 min in transfer buffer The water will wick up into the membrane wetting the entire surface If it is inserted too quickly into the water air gets trapped and will appear as white blotches in the membrane Protein will not transfer onto these areas This wetting procedure works for nitrocellulose and nylon membranes only PVDF mem branes are hydrophobic and will not wet simply from being placed into distilled water or transfer buffer For these membranes first immerse I to 2 sec in 100 methanol then equilibrate 10 to 15 min with transfer buffer Do not let membrane dry out at any time If this occurs wet once again with methanol and transfer buffer as described above 7 Moisten surface of gel with transfer buffer Place prewetted membrane directly on top side of gel i e anode side and remove all air bubbles as in step 5 Poor contact between the gel and membrane will cause a swirled pattern of transferred proteins on the membrane Some proteins will transfer as soon as the gel is placed on the membrane repositioning the gel or membrane can result in a smeared or double image on the developed blot The use o
330. oteins of 12 to 45 kDa Table 6 1 1 The stacking gel is the same regardless of the separating gel used 4 Using a Pasteur pipet apply the separating gel solution to the sandwich along an edge of one of the spacers until the height of the solution between the glass plates is 11 cm Use the solution immediately otherwise it will polymerize in the flask Sample volumes lt 10 ul do not require a stacking gel In this case cast the resolving gel as usual but extend the resolving gel into the comb step 10 to form the wells The proteins are then separated under the same conditions as used when a stacking gel is present Although this protocol works well with single concentration gels a gradient gel is recommended for maximum resolution see Alternate Protocol 5 5 Using another Pasteur pipet slowly cover the top of the gel with a layer 1 cm thick of H2O saturated isobutyl alcohol by gently layering the isobutyl alcohol against the edge of one and then the other of the spacers Be careful not to disturb the gel surface The overlay provides a barrier to oxygen which inhibits polymerization and allows a flat interface to form during gel formation The H20O saturated isobutyl alcohol is prepared by shaking isobutyl alcohol and H20 in a separatory funnel The aqueous lower phase is removed This procedure is repeated several times The final upper phase is H2O saturated isobutyl alcohol 6 Allow the gel to polymerize 30 to 60 m
331. own to 5 kDa To separate peptides below 5 kDa the tricine procedure must be modified by preparing a 16 5 T 2 7 C resolving gel that uses a 10 T spacer gel between the stacking Current Protocols in Cell Biology Molecular mass kDa 66 _ 45 36 29 24 20 1 14 2 JUL EIE Molecular mass kDa 205 116 97 4 66 45 29 Figure 6 1 9 Separation of membrane proteins by 5 1 to 20 5 T polyacrylamide gradient SDS PAGE Approximately 30 ul of 1x SDS sample buffer containing 30 ug of Alaskan pea Pisum sativum membrane proteins was loaded in wells of a 14 x 14 cm 0 75 mm thick gel Standard proteins were included in the outside lanes The gel was run at 4 mA for 15 hr and resolving gel Schagger and von Jagow 1987 The C is the percentage of cross linker bisacrylamide in the total monomer acrylamide plus bisacrylamide Continuous electrophoresis where the same buffer is used throughout the tank and gel is popular because of its versatility and simplicity The phosphate system described in Alternate Protocol3 is based on that of We ber et al 1972 Although unable to pro duce the high resolution separations of the dis continuous SDS PAGE procedures continu ous SDS PAGE uses fewer solutions with one basic buffer and no stacking gel Artifacts are also less likely to occur in continuous systems Pepsin for example migrates anomalous
332. p of each gel 14 Overlay lysis buffer with the degassed 0 1 M NaOH to fill the gel tubes Avoid mixing of NaOH with the lysis buffer 15 Pour the degassed 0 1 M NaOH into the upper chamber making sure that all the gel tubes are covered with the electrode solution Check carefully for leaks and air bubbles then place lid on apparatus 16 Connect the electrodes to a power supply by the red lead to the lower chamber and the black lead to the upper chamber The voltages and currents used during electrophoresis are dangerous and potentially lethal Safety considerations are given in the Electricity and Electrophoresis section of UNIT 6 1 17 Prefocus for 30 min using 500 V constant voltage Load the samples 18 Turn off power supply see Safety Considerations in uniT 6 1 disconnect leads and remove lid Using a 60 ml syringe remove the electrode solution 0 1 M NaOH from the upper chamber 19 Remove the electrode solution and the overlay solution from each tube Be careful not to damage the gel surface 20 Place 50 ul lysis buffer on the top of each gel Wait at least 2 min 21 Remove the lysis buffer from the tubes Rinsing the gels with lysis buffer removes any residual NaOH and protects the samples against exposure to high pH 22 Load protein samples to be analyzed and carefully overlay each sample with 50 ul lysis buffer diluted with water 8 2 v v Avoid mixing the buffer with the sample The overlay s
333. p to 1 month at 4 C Glycine gel buffer 4x 200 mM glycine pH 8 6 to 10 6 15 01 g glycine Add to 500 ml H O Adjust to pH 8 6 to 10 6 with 1 M NaOH Add H O to 1000 ml Store up to 1 month at 4 C Phosphate gel buffer 4x 400 mM sodium phosphate pH 5 8 to 8 0 55 2 g NaH PO H O Add to 500 ml H O Adjust to pH 5 8 to 8 0 with 1 M NaOH Add H O to 1000 ml Store up to 1 month at 4 C Current Protocols in Cell Biology Tris gel buffer 4x 200 mM Tris Cl pH 7 1 to 8 9 24 23 g Tris base Add to 500 ml H O Adjust to pH 7 1 to 8 9 with 1 M HCl Add H O to 1000 ml Store up to 1 month at 4 C Tris glycerol sample buffer 2x 25 ml 0 5 M Tris Cl pH 6 8 APPENDIX 24 20 ml glycerol 1 mg bromphenol blue Add H O to 100 ml and mix Store in 1 ml aliquots up to 6 months at 70 C Tris glycine electrophoresis buffer 15 1 g Tris base 72 0 g glycine H O to 5000 ml Store up to 1 month at 4 C COMMENTARY Background Information Under nondenaturing conditions in which protein activity native charge and conforma tion are sustained electrophoretic separation depends on many factors including size shape and charge Characteristics such as intrinsic molecular weight i e in the absence of denatu ration the number of isoforms and the pres ence of multimeric proteins can be determined with nondenaturing electrophoresis often called native electrophoresis The most important application of nonde naturing electrop
334. pe for 10x containing 1 w v SDS Denaturing BN transfer buffer see recipe Destaining solution see recipe Platform shaker PVDF polyvinylidene fluoride membrane see UNIT 6 2 Additional reagents and equipment for immunoblotting UNIT 6 2 1 Soak the first dimension BN gel in 1x PBS with 1 SDS for 15 min by shaking in a small tray on a platform shaker 2 Heat in a microwave at medium power until the solution boils Let the gel shake for another 10 min at room temperature 3 Transfer the denatured proteins to a PVDF membrane by wet or semi dry blotting UNIT 6 2 Use the denaturing BN transfer buffer since it includes SDS A protocol for wet transfer is described in UNIT 6 2 Basic Protocol 1 including Fig 6 2 1 Semi dry transfer is described in UNIT 6 2 Alternate Protocol 1 including Fig 6 2 2 Before transfer mark the ferritin marker with a pen on the membrane since it is difficult to see afterwards 4 Optional Partially remove the transferred Coomassie blue from the PVDF mem branes by incubation in destaining solution for 30 min Immunodetection can also be performed without the destaining step Coomassie blue cannot be removed completely and shows strong fluorescence at several wavelengths Therefore detection with fluorescently labeled antibodies cannot be done In this case a second dimension SDS PAGE has to be used for immunoanalysis Basic Protocol 2 5 Block the membrane and immunodetect under sta
335. pectrometry Nature 379 466 469 Wilson C M 1979 Studies and critique of amido black 10B Coomassie blue R and fast green FCF as stains for proteins after polyacrylamide gel electrophoresis Anal Biochem 96 263 278 Contributed by Esteban C Dell Angelica and Juan S Bonifacino National Institute of Child Health and Human Development National Institutes of Health Bethesda Maryland Current Protocols in Cell Biology Agarose Gel Electrophoresis of Proteins This unit describes the use of agarose gel as a matrix for the electrophoresis of proteins Although agarose is widely used as a material for molecular sieving it is not often used for the electrophoresis of proteins When it is used for this purpose it is generally employed for the electrophoresis of very large proteins In other electrophoresis proce dures it is part of a composite polyacrylamide agarose system The system described below utilizes agarose alone as the gel matrix As with acrylamide gels the proteins thus separated may be transferred to a membrane immunoblotting for further analysis or the proteins may be identified directly in the gel using stains or labeled antibodies A particular advantage of the method described in the Basic Protocol is the preparation of the gel in a horizontal electrophoresis bed which saves time and eliminates many of the difficulties commonly encountered in the preparation of acrylamide or composite gels in a vertical preparatio
336. plex Marker mixes 1 and 2 see recipe BN anode buffer see recipe Current Protocols in Cell Biology BN cathode buffer with 0 02 w v Coomassie blue see recipe BN cathode buffer with low 0 002 w v Coomassie see recipe Gel electrophoresis apparatus see UNIT 6 Gradient mixer self made or e g from BioRad Fig 6 1 2 Peristaltic pump Fig 6 1 2 Power supply Additional reagents and equipment for polyacrylamide gel electrophoresis UNIT 6 and protein staining in gels UNIT 6 6 Set up the apparatus 1 Wash glass plates and pouring devices extensively with water Do not use equipment that has been previously used for SDS PAGE No traces of detergent should be present on the glass plates 2 Assemble the glass plate sandwich of an electrophoresis apparatus UNIT 6 1 describes these procedures in detail Prepare the gradient 3 Set up the equipment to pour gradient gels as shown in Figure 6 1 2 UNIT 6 1 Gels will be poured at room temperature For reproducibility and to make the pouring more effective it is recommended to pour at least 10 gels at once using multicasting equipment e g BioRad 4 Close the valves and place a magnetic stir bar into the mixing chamber of the gradient maker Prepare low and high percentage BN separating gel solutions see Reagents and Solution Adjust the acrylamide bisacrylamide concentration of the high percentage BN separating gel solution according to your needs Table
337. pon the nature and the degree of glycosylation making it 100 fold more sensitive than the standard periodic acid Schiff base method using acidic fuchsin dye rosaniline The green fluorescent signal from Pro Q Emerald 300 stain can be visualized using a standard 300 nm UV UV B illumination source The Pro Q Emerald 488 Glycoprotein Blot Stain Kit is quite similar to the Pro Q Emerald 300 Glycoprotein Gel Stain Kit but is optimized for use with gel scanners equipped with 470 to 488 nm lasers The staining method is more reliable than mobility shift assays using glycosidases since even glycoproteins that are not susceptible to deglycosylation with specific enzymes may readily be identified as glycoproteins After detecting glycoproteins with Pro Q Emerald 300 dye total protein profiles may be detected using SYPRO Ruby protein blot stain SYPRO Ruby protein blot stain interacts noncovalently with basic amino acid residues in proteins The stain is capable of detecting lt 4 ng of protein band making it at least as sensitive as the best colloidal gold staining procedures available The orange red fluorescent signal from SYPRO Ruby protein blot stain can be visualized using a standard 300 nm UV UV B illumination source or alternatively may be excited using 470 to 488 nm laser gas discharge or xenon arc sources Materials Protein sample of interest PVDF membrane Fix solution see recipe Wash solution see recipe Pro Q Emerald 300 Gly
338. ponent of the macromolecule Packer et al 1999 Hirabayashi et al 2001 Until recently there have been relatively few methods available for the direct analysis of glycans on proteins transferred to membranes and most especially of glycans on proteins within polyacrylamide gels Packer et al 1999 Koketsu and Linhardt 2000 Raju 2000 Such methods could readily be incorporated into integrated proteomics platforms that utilize automated gel stainers image analysis workstations robotic spot excision instruments protein digestion work stations and mass spectrome ters Patton 2000a b There are two principal approaches to the detection of glycoproteins in gels and on blots reacting carbohydrate groups by periodate Schiff s base PAS chemistry and noncovalent binding of specific carbohydrate epitopes using lectin based detection systems The PAS method involves oxidation of carbohydrate groups followed by conjugation with a chromogenic substrate acid fuchsin Alcian Blue a fluorescent substrate dansyl hydra zine 8 aminonaphthalene 1 3 6 trisulfonate Pro Q Emerald dye biotin hydrazide or digoxigenin hydrazide Signal is detected directly in the case of the chromogenic or fluorescent conjugates and indirectly using enzyme conjugates of antibodies for bound digoxigenin or enzyme conjugates of streptavidin for bound biotin Lectins permit detection of certain structural subclasses of glycoproteins by similar methods to those used i
339. portant limitations of soluble ampholytes is the difficulty in obtaining highly reproducible pH profiles especially when very narrow pH ranges are needed An increasingly attractive alternative to soluble ampholytes is the use of immobilized pH gradient IPG gels see Basic Protocol 2 In this system the buffering side chains are covalently incorporated into the acrylamide matrix and any pH range and curve shape can be generated by pouring a gradient gel using two solutions that differ in ampholyte composition rather than acrylamide concentration As with tube gels the initial electro phoresis is followed by a second separation using SDS PAGE in a perpendicular direction see Basic Protocol 4 The use of IPG gels has recently increased for at least three major reasons many of the technical problems associated with their use have been solved or substantially minimized reproducible premade IPG gels are now commercially available and lately strong interest has arisen in using two dimensional gels for proteome analysis studies analyzing and comparing the complete protein profiles of cell lines tissue samples or single celled organisms Another common two dimensional electrophoresis format is a nonreducing reducing electrophoretic separation see Alternate Protocol 3 which provides useful information about intersubunit disulfides or protein protein complexes that have been cross linked using a bifunctional chemical cross linker containing a
340. posure time is difficult since the amount of radioactivity in the sample is often unknown A Geiger counter can offer some guidance with certain isotopes For highly ex posed film the length of time in developer can be reduced to produce a lighter image It is particularly important to remember that if ac curate quantification of the film image is de sired film must be preflashed so that there is a linear relationship between the amount of ra dioactivity in the sample and the image inten sity Artifacts such as black spots and stripes can be avoided during developing by making sure that no moisture comes in contact with the film and that films exposed at 70 C are brought to room temperature before developing Also it must be noted that B particles from weak iso topes such as 7H cannot penetrate plastic wrap and plastic wraps can attenuate signals from S and 4C up to two fold The protocols described here should yield a film image of a gel that can be quantified stored and photographed for publication Time Considerations Fixing a gel will require 45 min Drying will take an additional 2 hr for a gel 1 mm in thickness Incorporation of a fluor will add 45 min to the processing time For gels gt 1 5 mm thick or with gt 15 acry lamide an additional 30 min will be required for fixing and 30 additional minutes will be required for drying The length of exposure for films in autora diography can range from a
341. protein complexes and in some cases of intrasubunit disulfide bonds Both types of two dimensional gel electropho resis can be used for either analytical or preparative purposes Another useful method for electrophoretic separation of cellular proteins is one dimensional electrophoresis under nondenaturing conditions Two protocols describing variations of this method are included in unr 6 5 What distinguishes this method from those described in UNIT 6 1 and UNIT 6 4 is that protein samples are not exposed to denaturing agents i e SDS or urea either prior to or during electrophoresis Thus proteins migrate according to their native properties such as size shape and charge This allows analysis of the oligomeric state of proteins conformational changes charge heterogeneity and post translational modifications that affect conformation or charge while having minimal effects on the molecular weights of the proteins In many cases this method preserves the intrinsic function of the proteins which allows their detection with specific activity or binding assays The first protocol describes continuous electrophoresis on nondenaturing polyacrylamide gels This system involves electrophoresis on a single separating gel and uses the same buffer in the chambers and the gel The second protocol discontinuous electrophoresis on nondenaturing polyacrylamide gels is a variation of SDS PAGE in which SDS and reducing agents are omitted from all the soluti
342. proteins out of the IEF gel This short incubation before freezing will allow glycerol to diffuse into the gel Too short an incubation or agitation during freezing can result in gel breakage The total incubation time in equilibration buffer sum of the time prior to freezing and after thawing is critical and should be carefully controlled Insufficient incubation time in equilibration buffer will not allow sufficient time for SDS to diffuse into the gel and saturate sites on the proteins Excessive incubation times can result in appreciable protein losses due to diffusion out of the highly porous IEF gel CONDUCTING pH PROFILE MEASUREMENTS Standards with different isoelectric points can help in evaluating the performance of a specific system and determining the effective pH range in the isoelectric focusing gel Many pI standards are commercially available from different suppliers It is most useful to separate a mixture of standard proteins that is prepared from several individual proteins or purchased as a preformulated kit This mixture should be run in parallel with experi mental samples on a separate reference gel It is generally not recommended to run pI standards together in the same gel with samples because of possible interference with migration and identification of proteins of interest Instead of analyzing standard proteins a more precise evaluation of the pH profile can be made by directly measuring the pH throughout the gel using eithe
343. que uses isoelectric focusing IEF see Basic Protocols 1 and 2 followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS PAGE in a perpen dicular direction see Basic Protocol 3 This combination of isoelectric point pI and size separation is the most powerful tool for protein separations currently available After staining proteins appear on the final two dimensional gel as round or elliptical spots instead of the rectangular bands observed on one dimensional gels Although the total separating power of large format two dimensional gels is estimated to be gt 5000 spots per gel in practice a single two dimensional separation of a complex mixture such as a whole cell or tissue extract may produce 1000 to 2000 well resolved spots when a sensitive detection method is used Until recently the most common IEF procedures were based on the use of soluble ampholytes relatively small organic molecules with various isoelectric points and buff ering capacities The pH gradient for IEF gels is produced when the soluble ampholytes migrate in the gel matrix until they reach their isoelectric point Because stable pH gradients outside the pH 4 0 to 8 0 range are difficult to create with soluble ampholytes alternative protocols using nonequilibrium conditions are required to resolve proteins with pI values below 4 0 see Alternate Protocol 1 for acidic proteins or above 8 0 see Alternate Protocol 2 for basic proteins One of the more im
344. r 121 1 g Tris base 0 2 M final 500 ml H2O Adjust to pH 8 9 with concentrated HCl Dilute to 5 liters with H20 Store at 4 C up to 1 month Final concentration is 0 2 M Tris Cl pH 8 9 Cathode buffer 12 11 g Tris base 0 1 M final 17 92 g tricine 0 1 M final 1 g SDS 0 1 w v final recrystallization see recipe optional Dilute to 1 liter with H20 Do not adjust pH Store at 4 C up to 1 month Coomassie blue G 250 staining solution 200 ml acetic acid 20 v v final 1800 ml H2O 0 5 g Coomassie blue G 250 0 025 w v final Mix 1 hr and filter Whatman no paper Store at room temperature indefinitely Phosphate SDS electrophoresis buffer Dilute 500 ml of 4x phosphate SDS pH 7 2 Table 6 1 8 with H20 to 2 liters Store at 4 C up to 1 month Final concentrations are 0 1 M sodium phosphate pH 7 2 0 1 w v SDS Phosphate SDS sample buffer 2x for continuous systems 0 5 ml 4x phosphate SDS pH 7 2 Table 6 1 8 20 mM sodium phosphate final 0 2 g SDS 2 w v final recrystallization see recipe optional 0 1 mg bromphenol blue 0 001 w v final 0 31 g DTT 0 2 M final 2 0 ml glycerol 20 v v final Add H20 to 10 ml and mix Plug solution 0 125 M Tris Cl pH 8 8 APPENDIX 2A 50 w v sucrose 0 001 w v bromphenol blue Store at 4 C up to 1 month Recrystallized SDS optional High purity SDS is available from several suppliers but for some sensitive applica tions e g protein s
345. r narrower tubes the use of hydrostatic pressure is more appropriate see steps 3b to 7b below For long gels the needle can be extended by inserting a piece of capillary polyethylene tubing over the needle tip The amount of gel solution described in step 2 is sufficient for sixteen 3 mm tube gels that are 16 cm long 6a Immediately overlay each gel with 50 ul of 8 M urea A pipettor with a capillary pipet tip is a convenient tool for overlaying with urea Avoid mixing the overlay and gel solutions Polymerization starts to occur 15 min after the addition of TEMED and ammonium persulfate It is essential that the gels be poured and overlaid before significant polymerization has occurred 7a Let the gels polymerize at least 3 hr prior to use Urea decomposes at a substantial rate at room temperature therefore the gels should be used the same day they are cast Cast gels using hydrostatic pressure 3b Place a rubber band around the gel tubes so they form a tight bundle Place the bundle inside a larger glass cylinder that is sealed at the bottom with several layers of Parafilm All tubes must be precisely vertical The dimensions of the larger cylinder depend on the dimensions and number of gel tubes Excessive space will require more gel solution to cast the gels 4b Filter the gel solution using a 10 ml syringe and filter capsule Degas the gel solution briefly 5 min either with sonication or under vacuum Add 42 5 ul TEMED and
346. r Coomassie blue staining or electroblotting UNIT 6 2 for subsequent structural analysis a 50 ug total protein load should be sufficient for silver staining or immunoblotting and no less than 100 000 counts gel is recommended for proteins labeled with 3H C or S for autoradiography purposes Sample volumes should be lt 150 ul for 3 mm gels and lt 40 ul for 1 5 mm gels This implies at least a 5 ug ul protein concentration in the sample for gels to be stained with Coomassie blue Carefully fill all tubes with 0 1 M NaOH Avoid mixing the NaOH solution with the overlay solution and the sample Fill the upper reservoir with 0 1 M NaOH Be sure that all gel tubes are covered with the solution Run the gels 25 Connect the electrodes to a power supply with red to the lower chamber and black to the upper chamber 26 Focus for a total of 12 000 Vhr Unlike other electrophoretic techniques in IEF the volt hour is the most common unit describing the time of isoelectric focusing The initial voltage is usually set according to the desired number of volt hours in a way that is convenient for the operator i e so that the separation will run overnight but it should not be lt 400 V The upper voltage limit is restricted by heat released in the gels during isoelectric focusing At constant voltage the current will be the highest during the first hour of separation The initial current will be strongly influenced by the ionic s
347. r a surface pH electrode or the following procedure 1 Prepare and focus one or two gels see Basic Protocol 1 steps 1 to 26 without any sample in parallel with experimental samples 2 Prepare 20 to 40 glass test tubes each containing ml high purity degassed water for each gel that will be used to measure the pH gradient measurements on duplicate gels are recommended The number of tubes required per gel equals twice the gel length in cm 3 After electric focusing is completed extrude the blank gels see Basic Protocol 1 steps 27 to 29 Briefly rinse the gels with water After extrusion gel surfaces may be contaminated with electrode solutions Rinsing with water is essential for obtaining reliable pH profiles 4 Place the gel on a glass plate with a plastic ruler below the plate Cut the gel into 0 5 cm pieces using a sharp razor blade 5 Place each gel piece in a test tube containing ml water Do not mix the order of samples because each gel piece represents a single pH profile data point 6 Place all test tubes on a shaker and shake gently for 1 hr at room temperature Current Protocols in Cell Biology SUPPORT PROTOCOL 1 Electrophoresis and Immunoblotting 6 4 7 Supplement 4 ALTERNATE PROTOCOL 1 ALTERNATE PROTOCOL 2 Two Dimensional Gel Electrophoresis 6 4 8 Supplement 4 7 Read the pH of each solution and plot the pH profile as a function of the distance from the top
348. r exposure to conditions that activate the vWF protease lane 2 Paired samples from patients with thrombotic thrombocytopenic purpura who have an inhibitor to the vWF protease are seen in lanes 3 to 8 Odd lanes contain plasma samples using conditions that do not activate the vWF protease and even lanes contain the paired sample that was exposed to conditions that activate the protease Very little proteolysis is observed in these patients plasmas due to the presence of an inhibitor even lanes The numbers above the patient lanes indicate the retention of the high molecular weight multimers tion procedures can be completed the next day The Alternate Protocol takes up to 5 or more days to obtain results largely due to the time required for the incubation with antibody and development of the autoradiogram Also a ra diolabeled antibody specific for the protein to be identified must be available Literature Cited Aronson D L Krizek D M and Rick M E 2001 A rapid assay for the vWF protease Thrombosis and Hemostasis 85 184 185 Hoyer L W and Shainoff J R 1980 Factor VII related protein circulates in normal human plasma as high molecular weight multimers Blood 55 1056 1059 Krizek D M and Rick M E 2000 A rapid method to visualize von Willebrand factor multimers using agarose gel electrophoresis immunolo calization and luminographic detection Throm bosis Research 97 457 62 Krizek D M and Rick M E 2001 C
349. r support GelBond provides a prac tical way of casting running and staining ex tremely thin gels When gels lt 0 75 mm thick are used reagents have much better access both into and out of the gel reducing staining time in both Coomassie blue and silver stain ing Double and broadened images caused by differential migration of the protein across the thickness of the gel are minimized improving resolution Critical Parameters and Troubleshooting If an electrophoretically separated protein will be electroeluted or electroblotted for sequence analysis the highest purity reagents available should be used If necessary SDS Electrophoresis an Immunoblotting 6 1 35 Supplement 37 One Dimensional SDS PAGE 6 1 36 Supplement 37 can be purified by recrystallization following the procedure given in Reagents and Solutions If the gels polymerize too fast the amount of ammonium persulfate should be reduced by one third to one half If the gels polymerize too slowly or fail to polymerize all the way to the top use fresh ammonium persulfate or increase the amount of ammonium persulfate by one third to one half The overlay should be added slowly down the spacer edge to prevent the overlay solution from crashing down and disturbing the gel interface After a separating gel is poured it may be stored with an overlay of the same buffer used in the gel Immediately prior to use the stack ing gel should be poured
350. r the stacking gel instead of the SDS containing counterparts Table 6 1 1 Prepare a minimum of four separate gels at different acrylamide concentra tions A typical range of concentrations is from 5 to 12 5 e g 5 7 5 10 12 5 acrylamide As with SDS PAGE typical gel thickness ranges from 0 75 to 1 5 mm The 0 75 mm thick gels are recommended because they offer a combination of fast staining and high resolution Mix protein sample of interest 1 1 with 2x Tris glycerol sample buffer to attain a 1 to 2 ug ul final concentration Also prepare native protein standards Remove comb rinse wells and load 10 to 20 ul per well for Coomassie brilliant blue staining and 1 to 2 ul for silver staining Some proteins must be dissolved in 50 mM NaCl or water to become fully solubilized prior to mixing with the sample buffer Sigma 1986 Assemble gel electrophoresis unit using Tris glycine electrophoresis buffer to fill both lower and upper buffer chambers Connect power supply and conduct electro phoresis Conditions for separation are the same as for discontinuous SDS PAGE i e 30 mA for 1 5 mm thick gels 15 mA for 0 75 mm thick gels For standard size gels the separation takes 4 to 5 hr for minigels 1 to 2 hr is required Alternatively standard gels can be run at 4 to 6 mA gel overnight After the bromphenol blue R marker has reached the bottom of the gel fix and stain the proteins in the gels according to APPEND
351. ra tion distances shorter than the spatial resolution will fail to provide reliable data Many factors including the amount of sample loaded gel pore size buffer constituents and electropho resis field strengths can dramatically affect separation and resolution of biomolecules Likewise detection methods that can only gen erate a small range of discrete intensity values will not benefit from systems with improved intensity resolution IMAGE CAPTURE Devices Capturing digital images involves a detec tion beam or source a sensor for that beam or source and some method of assembling a two dimensional image from the data generated Most systems use a light source for detection The light wavelengths used range from ultra violet UV to infrared IR and can be broad spectrum or narrow wavelength Broad spec trum detection is more versatile since it can often be used for more than one detection wave length However when compared to narrow wavelength sources such as lasers broad spec trum detection suffers from reduced sensitivity and reduced dynamic range While many types of light sensors have been used including charge coupled devices CCDs charge injec tion devices CIDs and photon multiplier tubes PMTs technology advances in CCDs have led to their dominance CCDs are semi conductor imaging devices that convert pho tons into charge This charge is then read and converted into a digital format via an analog t
352. re agent related problems are encountered the final stained two dimensional gels should contain nu merous rounded or slightly elliptical spots Typi cally more than 1000 spots can be detected on a standard 16 x 14 cm gel when using a sensitive staining protocol such as silver staining or autora diography and a whole cell extract as a sample Some horizontal streaks for most high molecu lar mass proteins proteins exceeding 100 kDa are common owing to the decreased solubility of larger proteins near their isoelectric points even in the presence of urea and nonionic detergent However excessive horizontal smearing of pro teins smaller than 100 kDa indicates poor isoelec tric focusing which could be related to one or more of the following factors sample improperly solubilized or contaminated with interfering sub stances such as large nucleic acid molecules poor purity of reagents check the urea first poor quality ampholytes or insufficient isoelectric fo cusing total volt hours too low It is important to note that in general the solubility ofany protein is the lowest near its isoelectric point but there are vast differences among proteins in terms of both the minimum concentration where precipi tation becomes a problem and the degree to which precipitation can be prevented by adding different solubilization agents The best conditions for maintaining solubility during isoelectric focusing for a given sample type must be
353. re pare fresh buffer and acrylamide monomer stocks If the protein bands are diffuse in crease the current by 25 to 50 to complete the run more quickly and minimize band diffu sion use a higher percentage of acrylamide or try a gradient gel Lengthy separations using gradient gels generally produce good results Fig 6 1 9 Check for possible proteolytic degradation that may cause loss of high molecular weight bands and create a smeared banding pattern If there is vertical streaking of protein bands decrease the amount of sample loaded on the gel further purify the protein of interest to reduce the amount of contaminating pro tein applied to the gel or reduce the current by 25 Another cause of vertical streaking of protein bands is precipitation which can sometimes be eliminated by centrifuging the sample or by reducing the percentage of acry lamide in the gel Proteins can migrate faster or slower than their actual molecular weight would indicate Abnormal migration is usually associated with a high proportion of basic or charged amino acids Takano et al 1988 Other problems can occur during isolation and preparation of the protein sample for electrophoresis Pro teolysis of proteins during cell fractionation by endogenous proteases can cause subtle band splitting and smearing in the resulting electrophoretogram electrophoresis pattern Many endogenous proteases are very active in SDS sample buffers and will rap
354. re that provides basic im age analysis tools for the Macintosh http www inforamp net poynton Poynton color html Contains an excellent description of gamma correc tion in the Gamma FAQ http www Immb ncifcrf gov EP table2Ddatabases html A list of links to many two dimensional databases that are available via the Internet Contributed by Scott Medberry Amersham Pharmacia Biotech San Francisco California Sean Gallagher Motorola Corporation Tempe Arizona Current Protocols in Cell Biology Two Dimensional Blue Native Polyacrylamide Gel Electrophoresis Wolfgang W A Schamel Max Planck Institut fiir Immunbiologie und Universitat Freiburg Biologie III Freiburg Germany ABSTRACT Multiprotein complexes play crucial roles in nearly all cell biological processes Blue Native Polyacrylamide Gel Electrophoresis BN PAGE is a powerful method to study these complexes It is a native protein separation method that relies on the dye Coomassie blue to confer negative charge for separation It has a higher resolution than gel filtration or sucrose density ultracentrifugation and can be used for protein complexes from 10 kDa to 10 MDa If a second dimension SDS PAGE is applied two dimensional BN SDS PAGE the size subunit composition and relative abundance of the different multiprotein complexes can be studied In recent years there has been a large increase in the number of publications where BN PAGE was used to study pro
355. resis of Proteins 6 7 6 Supplement 15 glass plate GelBond film spacer plate with 0 5 mm spacers flexiclamps Figure 6 7 1 Diagram of casting gel in the Alternate Protocol The layers include back to front the glass plate the larger of the two plates GelBond film hydrophilic side adherent to the glass plate and the spacer plate with attached 0 5 mm spacer bars The agarose fills the narrow space between the GelBond and the spacer plate 12 After filling lay the plate assembly flat and allow to cool and solidify 45 min Electrophorese gel 13 14 15 16 Position the Gelman Delux electrophoresis chamber or equivalent electrophoresis apparatus on a flat surface and fill each electrode chamber with 400 to 450 ml agarose running buffer Place 104 x 253 mm paper electrophoresis electrode wicks in each chamber to equilibrate in running buffer Remove the flexiclamps Insert a thin spatula blade between the spacer plate and the gel attached to the glass plate Carefully pry upward to separate the spacer plate Examine the gel against a bright light for bubbles thin areas or areas of separation from the GelBond film The agarose gel should now be attached to the GelBond support backing and be easily handled Punch the required number of 0 1 x 10 mm wells using a flattened no 2 cork borer Use a fine set of forceps to remove the agarose from the interi
356. rest because Coomassie blue interferes with fluorescence based visualization methods and because native proteins are often detected by different antibodies than those that detect denatured proteins A powerful method to determine the stoichiometry of multiprotein complexes is the Native Antibody based MObility Shift NAMOS assay see Basic Protocol 3 It is based on one dimensional BN PAGE and uses the fact that proteins migrate more slowly during the electrophoresis when an anti subunit antibody is bound NOTE High purity water e g Milli Q or distilled water should be used for all solutions For cautions relating to electricity and electrophoresis see Safety Considerations in the introduction to UNIT 6 1 NOTE Wear powder free gloves throughout the procedure and work on ice or at 4 C whenever native proteins protein complexes are present CAUTION Acrylamide is hazardous see APPENDIX 2A for guidelines on handling storage and disposal FIRST DIMENSION BLUE NATIVE ELECTROPHORESIS In this protocol the pouring and running of vertical slab blue native BN gels is described BN gels are gradient gels of low acrylamide percentage and strength Thus pouring and handling of the gels is not trivial The gradient has to be even in order to prevent any step that would result in the erroneous accumulation of proteins at a particular height of the gel The BN gel solution with the higher acrylamide bisacrylamide concentration is heavier th
357. ride PVDF membrane Millipore Additional reagents and equipment for protein transfer to membranes and immunoblotting UNIT 6 2 Cast agarose gels 1 Weigh 1 2 g SeakKem HGT P agarose and transfer to a 250 ml flask containing 200 ml of 1x electrophoresis buffer Add a Teflon coated magnetic stir bar and tightly cover the flask with aluminum foil Heat in a boiling water bath with slow mixing to avoid bubbles until clear Alterna tively microwave until boiling CAUTION Whenever a solution is heated in a microwave the chance of superheating boil up is always present Protective gloves gown and eyewear should be worn at all times Assemble Horizon 20 25 horizontal electrophoresis apparatus or equivalent accord ing to manufacturer s recommendations The wedge shaped casting dams of the Horizon Life Technologies horizontal apparatus are easily placed in backwards resulting in leaking of liquid agarose Care should be exercised so that the dams form a perpendicular angle with the UVT tray Cool agarose to 55 to 60 C and pour the molten agarose into the electrophoresis apparatus to a depth of 4 mm It is often helpful to prewarm the electrophoresis apparatus with warm water or in an oven to avoid cooling the agarose and causing the agarose to solidify unevenly Carefully insert the desired e g 1 x 9 mm 20 well Teflon comb with care to avoid bubbles Current Protocols in Cell Biology Remo
358. rientation of electrodes later in the procedure anode side cut corner anode side anode corner cut by manufacturer A A cut in this direction 4 cathode side Figure 6 4 1 Marking orientation of a precast IPG gel when only a portion of the gel is used Current Protocols in Cell Biology Use the Reswelling Cassette to rehydrate the gel Connect silicone tubing through hole in the bottom corner of the U frame plate seal with silicone glue and connect the pinchcock to the other end of the tubing Place a glass plate on a clean flat surface and wet with a few drops of water Place the gel on the plate gel side up Gently roll with a clean rubber roller to remove any air bubbles Cover the plate and gel with the plate fitted with the U frame The U frame plate should be coated with a thin layer of Repel Silane to prevent the gel from sticking to the plate Place clamps around the edges of the plates making sure the seal is tight Slowly fill the cassette with the desired rehydration solution using a 20 ml syringe connected to the silicone tubing and let stand for the recommended amount of time Precool electrophoresis unit 1 to 2 hr prior to electrophoresis see step 9 Reswelling with water for 2 to 3 hr is normally sufficient If using additives such as urea Triton glycerol or reducing agents allow the gel to rehydrate overnight Additives can be used to improve solubility of protein
359. ris Cl SDS pH 6 8 Table 6 1 1 20 ml glycerol 20 final 4 g SDS 4 w v final recrystallization see recipe optional 2 ml 2 ME or 3 1 g DTT 0 2 v v 2 ME or 0 2 M DTT final 1 mg bromphenol blue 0 001 w v final Add H20 to 100 ml and mix Store in 1 ml aliquots at 70 C To avoid reducing proteins to subunits if desired omit 2 ME or DTT reducing agent and add 10 mM iodoacetamide to prevent disulfide interchange SDS sample buffer 6x for discontinuous systems 7 ml 4x Tris Cl SDS pH 6 8 Table 6 1 1 3 0 ml glycerol 30 v v final 1 g SDS 10 w v final recrystallization see recipe optional 0 93 g DTT 0 6 M final 1 2 mg bromphenol blue 0 012 w v final Add H20 to 10 ml if needed Store in 0 5 ml aliquots at 70 C Tricine sample buffer 2x 2 ml 4x Tris Cl SDS pH 6 8 Table 6 1 1 0 1 M 2 4 ml 3 0 g glycerol 24 v v final 0 8 g SDS 8 w v final recrystallization see recipe optional 0 31 g DTT 0 2 M final 2 mg Coomassie blue G 250 0 02 w v final Add H20 to 10 ml and mix Electrophoresis and Immunoblotting al 6 1 31 Current Protocols in Cell Biology Supplement 37 One Dimensional SDS PAGE 6 1 32 Supplement 37 COMMENTARY Background Information Although electrophoresis has been studied for over two centuries Arne Tiselius Univer sity of Uppsala Sweden put the technique on the map with his Ph D thesis in 1930 which demonstrated moving zones of ser
360. rm environments The versatil ity of TIFF can also be a weakness Since there are many different tagging schemes and since not all programs support all possible compres sion and color schemes it is sometimes not possible for one program to access the infor mation in a TIFF file generated by a different program GIF Graphics Interchange Format is a file format that is widely encountered on the In ternet due to its compactness and stand ardization Its compactness is attributable to a mandatory modified LZW compression An other feature of GIF is the use of a LUT to index the values in the image One interesting ability of GIF is that it supports storing multiple im ages within a single file This can offer some advantages for applications such as time lapse image capture A GIF image can contain no more than 256 individual color or gray levels and does not support intensity resolutions higher than 8 bit In addition since the image Electrophoresis and Immunoblotting 6 9 7 Supplement 16 Digital Electrophoresis Analysis 6 9 8 Supplement 16 is implemented as a LUT it also is not a true gray scale image Due to these limitations and others alternative formats such as PNG have been developed to replace GIF PICT is a file format and graphics metafile language it contains commands that can be played back to recreate an image designed for the Apple Macintosh It can contain both bit map images and vec
361. rom 5 to 15 acrylamide and 0 2 to 0 5 bisacrylamide see Table 6 1 1 The relation ship between the relative mobility and log molecular weight is linear over these ranges Fig 6 1 6 With the use of plots like those shown in Figure 6 1 6 the molecular weight of an unknown protein or its subunits may be determined by comparison with known pro tein standards Table 6 1 2 In general all of the procedures in this unit are suitable for ra diolabeled and biotinylated proteins without modification Basic Protocol 1 relies on denaturing pro teins in the presence of SDS and 2 ME or DTT Under these conditions the subunits of proteins are dissociated and their biological activities are lost A true estimate of a pro tein s molecular size can be made by com paring the relative mobility of the unknown protein to proteins in a calibration mixture Support Protocol 4 Gradient gels Alternate Protocol 5 simplify molecular weight deter minations by producing a linear relationship between log molecular weight of the protein and log T over a much wider size range than single concentration gels Although per cent acrylamide monomer is a more common measure of gel concentration T the per centage of total monomer acrylamide plus bisacrylamide in the solution or gel is used for molecular weight calculations in gradient gels The T of a stained protein is estimated assuming the acrylamide gradient is linear For example pro
362. ropriate This method requires a larger excess of reagents and special casting cylinders Many types of ampholytes are readily available from different suppliers to form the desired pH profiles As ampholytes may vary significantly in their performance careful selection of the appropriate ampholytes is usually necessary see Commentary Materials Chromic acid in acid resistant container Urea ultrapure 30 acrylamide 0 8 bisacrylamide see recipe 20 w v Triton X 100 see recipe Ampholytes e g pH 3 10 2D ESA TEMED N N N N tetramethylethylenediamine 2 5 w v ammonium persulfate see recipe prepare immediately before use 8 M urea see recipe prepare immediately before use 0 1 M orthophosphoric acid H PO see recipe 0 1 M NaOH make fresh daily Lysis buffer see recipe Protein samples to be analyzed Equilibration buffer see recipe 2 Mercaptoethanol Isoelectric focusing apparatus e g Protean II xi 2D from Bio Rad or equivalent with glass tubes casting stand buffer chambers rubber grommets and plugs 37 C water bath continued Current Protocols in Cell Biology 110 C oven 10 ml syringe equipped with filter capsule 0 22 or 0 45 um e g Costar uStar LB 10 ml syringe equipped with blunt needle e g 20 G x 6 in 15 cm or 18 G x 6 in 15 cm Large glass cylinder sealed at bottom with Parafilm optional for hydrostatic pressure casting method only 2000 V power supply 60 ml syri
363. roteins from polyacrylamide gels for amino acid sequence analysis Methods Enzy mol 91 227 236 Laemmli U K 1970 Cleavage of structural pro teins during the assembly of the head of bacte riophage T4 Nature 227 680 685 Matsudaira P T and Burgess D R 1978 SDS mi croslab linear gradient polyacrylamide gel elec trophoresis Anal Biochem 87 386 396 Okajima T Tanabe T and Yasuda T 1993 Nonurea sodium dodecyl sulfate polyacrylamide gel electrophoresis with Electrophoresis and Immunoblotting 6 1 37 Supplement 37 One Dimensional SDS PAGE 6 1 38 Supplement 37 high molarity buffers for the separation of proteins and peptides Anal Biochem 211 293 300 Ploegh H L 1995 One Dimensional Isoelectric Focusing of Proteins in Slab Gels Jn Current Protocols in Protein Science J E Coligan B M Dunn D W Speicher and P T Wingfield eds pp 10 2 1 10 2 8 John Wiley amp Sons Hoboken NJ Schagger H and von Jagow G 1987 Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa Anal Biochem 166 368 379 Takano E Maki M Mori H Hatanaka N Marti T Titani K Kannagi R Ooi T and Murachi T 1988 Pig heart calpastatin Iden tification of repetitive domain structures and anomalous behavior in polyacrylamide gel elec trophoresis Biochemistry 27 1964 1972 Weber K Pringle
364. round value for each pixel in a band For absorptively detected bands intensity values are converted to OD values The total value that is calculated is equivalent to the volume of the band and can be directly compared to other bands that are within the linear range for the visualization method If standards of known amounts are loaded onto the same gel they can be used to generate a standard curve that converts band volume into standard units such as micrograms For greatest accuracy it is important to be able to generate multiple standard curves when us ing visualization methods such as Coomassie blue staining that are affected by band or spot composition Quantitation becomes more complicated when bands are not fully resolved In this case material from one band is contributing to the volume measurement of an adjacent band and vice versa The simplest method for handling this is to partition into each band only the volume within its edges Alternatively a Gauss ian curve can be fitted to each band and the volume contained within the curve used to estimate the amount of the band Since most electrophoresis bands have a pronounced skew towards the leading edge of the band modified Gaussian curves have also been used Smith and Thomas 1990 In either case the curve fitting process is calculation intensive and can significantly increase analysis times for images with many bands Software for Two Dimensional Analysis In two d
365. rrent Protocols in Cell Biology BASIC PROTOCOL 3 Electrophoresis and Immunoblotting 6 6 5 Supplement 6 l NOTE All steps should be performed at room temperature Remove polyacrylamide gel from electrophoresis assembly and place it in a plastic container with lid containing a large excess 10 gel vol of SYPRO Ruby protein gel stain Incubate 3 hr with gentle agitation The use of a platform shaker is recommended Remove the SYPRO Ruby protein gel stain and rinse the gel briefly with distilled water Do not reuse the stain solution Follow local recommendations for the disposal of poten tially toxic organic compounds Add 10 gel vol distilled water and incubate 10 min with gentle agitation Change the distilled water in the container and incubate another 10 min with gentle agitation Visualize protein bands by fluorescence on a 300 nm UV transillumination unit Images can be acquired using a photographic camera with appropriate film e g Polaroid 667 black and white print film or a CCD camera CAUTION Use UV rated eye protection to avoid direct exposure of the eyes to the UV light At this point the gel is ready for downstream applications e g electroelution and immunoblotting BASIC REVERSIBLE PROTEIN STAINING WITH ZINC PROTOCOL 4 Staining Proteins in Gels 6 6 6 Supplement 6 2 In this method protein bands separated on SDS PAGE gels one or two dimensio
366. rrent Protocols in Cell Biology Supplement 6 Table 6 6 3 Troubleshooting Guide for Fluorescent Staining Basic Protocol 3 Problem Cause Solution Protein bands are absent or too faint Clear background Highly fluorescent background Heavy stain at the SDS PAGE migration front Odd marks at the gel surface Amount of protein s below detection limit Direct eye visualization is not sensitive enough Photographic or imaging conditions are not appropiate Stain solution too old or inactivated by light Protein s of interest pre stained or containing colored prosthetic groups Too much unbound dye remains in the gel matrix Gel is placed for visualization on an autofluorescent plastic surface e g Saran Wrap Gel is attached to a polyester surface with affinity for the dye e g backing material of PhastGels SYPRO dyes bind micellar SDS at the migration front Portions of the gel squeezed or have powder deposits due to handling Check protein concentration in original sample Stain the gel with silver Basic Protocol 2 Visualize the bands by using a CCD camera or by photography Use sensitive photographic films Polaroid 667 black and white or similar film or appropiate CCD camera Increase exposure integration time Use fresh stain solution Protect it from the light Do not reuse it If possible remove prosthetic group s from proteins and avoid using pre stained proteins Otherwise use an
367. s called the trailing ions whose electrophoretic mobility is less than the mobility of the pro teins in the sample The net result is that the faster migrating ions leave a zone of lower conductivity between themselves and the mi grating protein The higher voltage gradient in this zone allows the proteins to move faster and to stack in the zone between the leading Current Protocols in Cell Biology Table 6 1 12 Key Applications of Protein Electrophoresis For more Technique Separation principle Application q p P P PP information Native PAGE Native charge size Purification determination of UNIT 6 5 and shape native protein size and identification of protein complexes 1 D SDS PAGE Size dependent SDS Size estimation purity check UNIT 6 1 imparts a negative purification subunit charge to proteins composition protein giving a constant expression charge to mass ratio IEF Intrinsic charge with Purification purity check Ploegh 1995 both native and isoenzyme analysis denatured proteins 2 D SDS PAGE Isoelectric point inthe Protein expression UNIT 6 4 first dimension size in purification posttranslational the second analysis proteomics a Abbreviations 1 D one dimensional 2 D two dimensional IEF isoelectric focusing CH Ox UNH UNH D HoN Zz re 0 CH 0 CHa HC N N methylenebisacrylamide acrylamide acrylamide 0 NH O n S fe NH HN n 0 polyacrylamide S 0 7 persulfate 2 S
368. s that are used as seeds for subsequent matches Appel et al 1991 Monardo et al 1994 There are many methods for finding the land marks in both images including finding the highest intensity spots finding spots in unique clusters and manual positioning The most common procedure from this point is to derive a vector that describes the direction and extent of the path from one matched spot to the other when the two images are superimposed The vector is used as the basis for finding more matches near the landmark matches To allow Electrophoresis and Immunoblotting 6 9 11 Supplement 16 Coefficient Dice Algorithm UPGMA Listeria serotype 4b MS ee we T Listeria serotype 4c MPP TT Unknown 7 was ss Unknown 10 wis 9 Unknown 1 Listeria serotype 4d eT Listeria serotype 4e eT Listeria serotype 4a Sw Listeria serotype 1 als FF Ff Listeria serotype 3 E E F Listeria monotype B Listeria serotype 2 E TF FF Unknown 11 T a A Unknown 12 _ a E Unknown 3 Unknown 4 T a Unknown 5 _ E A Unknown 9 T E Unknown 2 o Listeria monotype A SS F T Unknown 6 Ps ws F 0 45 Unknown 8 a E 05 06 07 08 O89 Figure 6 9 3 Example of a dendrogram generated from similarity data on band matching between lanes DNA samples from 22 isolates of Listeria were subjected to Random Amplification of Polymorphic DNA RAPD analysis and the resulting electrophoresis image was analyzed with ImageMast
369. s near their isoelectric point Reducing reagents such as DTT are used to reduce disulfide bonds When gel has been allowed to rehydrate completely remove the clamps and gently pry the plates apart Moisten a piece of filter paper with water and place on top of the gel then layer with a piece of dry filter paper Gently blot the gel by rolling over the dry filter paper with the rubber roller to remove excess water The gel is now ready to be placed on the cooling plate Do not let the gel dehydrate prior to placing it on the cooling plate in step 11 Run the gel 9 10 11 12 13 14 Connect the flatbed electrophoresis unit to a recirculating cooling water bath Allow to cool to 10 C for 1 to 2 hr to ensure even cooling If the gel has been rehydrated in the presence of urea do not cool below 15 C so that urea does not precipitate Pipet 2 to 3 ml paraffin oil onto the surface of the cooling plate Position the gel on the cooling plate being careful not to trap air bubbles between the gel and the plate Orient the gel so that the polarity of the gel matches the polarity of the cooling plate Soak two electrode strips with 3 ml water then blot to remove excess water Lay a blotted electrode strip along each long edge of the gel Cut off the ends of the electrode strip so that it does not extend beyond the edge of the gel Load protein samples to be analyzed onto the gel Use an applicator strip for sa
370. s occurs it is possible to calibrate one image and then pass the calibration information via the matches to the related images Quantity determination is similar in many regards to that which occurs in one dimen sional analysis but there are some differences If spot edges are detected a simple method of determining spot volume is to take the sum of the intensity value of each pixel in the spot reduced by a background intensity value Mul tiple Gaussian curves can also be fitted to the spot to approximate the volume Garrels 1989 More difficult is attempting to compen sate for a skewed distribution in a size separat ing dimension while trying to use a regular Gaussian fit for a pI separation such as is encountered with the most common form of two dimensional protein separations The dis tribution of background makes quantitation more difficult in two dimensional gels There is no lane dependent component so it is nec Current Protocols in Cell Biology essary to use other methods such as image stripes finding local minimum values or using values derived from the spot edges to determine background values Matching Matching is the process in which proteins or nucleic acids with similar separation properties are linked or clustered together Matching can occur within one image or between multiple images as long as a frame of reference is estab lished Matching allows for comparisons be tween samples It also makes annotatio
371. s of an anti X Fab fragment were incubated with the lysate before separation Consequently a new band band b appeared with a concomitant loss of band a lanes 2 to 6 Band b corresponded to a Fab XY2 complex indicating that XY contained one binding site for the anti X Fab fragment and thus one copy of protein X An anti Y Fab fragment produced two bands lanes 8 to 12 bands b and c demonstrating that XY2 had two copies of protein Y In B complete antibodies were used for the NAMOS assay In contrast to the Fab fragment an antibody is able to simultaneously bind to two XY2 complexes Using anti Y one can see that these super complexes band d disappeared with increasing concentrations of the antibody lanes 2 to 5 At saturating concentrations all XY2 complexes bound to two antibodies lanes 5 and 6 band c indicative of two copies of protein Y in the complex Thus complete antibodies also allow the determination of stoichiometry As a control an irrelevant anti Z antibody did not produce any shift of complex XYo lanes 7 to 12 A similar wet experiment and possible difficulties in performing the NAMOS assay are described in Swamy et al 2007 Current Protocols in Cell Biology Materials Monoclonal antibodies that react with the proteins of interest BN dialysis buffer see recipe Sample for BN PAGE 1 ml reaction tubes e g microcentrifuge tubes Additional reagents and equipment for casting and running BN PAGE gels
372. s to protocols that include nonionic detergents and urea see Basic Protocol 1 In addition the presence of DNA and RNA in crude cell extracts further complicates isoelectric focusing The protocol presented below is suitable for preparing samples from cell cultures and is based on quantities compatible with silver staining or Coomassie blue staining If smaller cell numbers and high sensitivity detection methods such as autoradiography are used volumes and quantities should be adjusted as needed In this protocol the cells are harvested and washed in phosphate buffered saline PBS with proteolysis inhibitors then lysed in Tris SDS buffer using sonication after which the total protein concentration is determined in the lysate The lysate is further treated with a mixture of DNase and RNase and additional SDS and reducing agent are added At this stage the samples can be stored at 80 C for an extended time or after addition of urea and lysis buffer may be loaded directly onto prefocused IEF gels Filtration of the final sample prior to loading onto the IEF gel is essential for quality of isoelectric focusing Current Protocols in Cell Biology SUPPORT PROTOCOL 4 Electrophoresis and Immunoblotting 6 4 17 Supplement 4 Materials Cell culture flasks containing cells of interest PBS with proteolysis inhibitors PBS I buffer see recipe Dry ice ethanol optional for freezing samples Tris SDS buffer see recipe BCA
373. s using other glycopro tein detection methods This avoids erroneous interpretation of results arising from low levels of noncovalent binding of dye molecules or confusion arising from the inherent fluores cence of certain high abundance proteins in the gel profile The most common problem encountered using the Pro Q glycoprotein detection kit with concanavalin A is poor signal intensity This is usually due to decomposition of the DDAO phosphate stock solution When the stock solu tion appears by eye to be a blue color the substrate has broken down and is no longer usable Current Protocols in Cell Biology Table 6 8 1 Membranes Comparison of Commonly Used Glycoprotein Detection Methods for Polyacrylamide Gels and Electroblot Time Detection sensitivity 1 ol acid glycoprotein 40 CHO Detection method required ee 2 glucose oxidase 12 CHO Assets or liabilities hr PS 3 avidin 7 CHO Gels Blots Acid fuchsin sulfite 5 6 7 1 75 ng 20 ng short procedure pararosaniline 2 150 ng 75 ng can use either on blots 3 150 ng 75 ng or in gels poor sensitivity Biotin hydrazide 6 11 1 na 18 37 ng signal fades over time streptavidin HRP 2 na 37 ng optimal image 20 30 min Luminol detection 3 na 150 ng cannot save and reimage reagents blots Biotin hydrazide 5 6 11 1 na 2 ng good sensitivity streptavidin alkaline 2 na 5 9 ng can save and reimage phosphatase
374. sensitivity with minimum background can be complex and the manufacturer s instructions should be consulted see Reagents and Solutions The procedure described in Alternate Protocol 4 gives reasonable sensitivity on nitrocellulose PVDF and nylon membranes with a minimum of steps Critical Parameters First and foremost the antibody being used should recognize denatured antigen Nonspe cific binding of antibodies can occur so control antigens and antibodies should always be run in parallel Time of transfer and primary anti Current Protocols in Cell Biology body and conjugate dilutions should always be optimized A variety of agents are currently used to block binding sites on the membrane after blot ting Harlow and Lane 1988 These include Tween 20 PVP nonfat dry milk casein BSA and serum A 0 1 v v solution of Tween 20 in TBS TTBS a convenient alternative to protein based blocking agents is recom mended for chromogenic development of ni trocellulose and PVDF membranes Blake et al 1984 In contrast to dry milk TBS blocking solution BLOTTO TTBS is stable and has a long shelf life at 4 C Furthermore TTBS gen erally produces a clean background and permits subsequent staining with India ink However even with the application of such standard blocking procedures as 5 to 10 milk protein or 0 05 to 0 1 Tween 20 background can still be a significant problem If this happens using a blocking protei
375. seradish peroxidase HRPO based immunodetection procedures while BCIP NBT is recommended for alkaline phosphatase AP based procedures see Table 6 2 1 After incubation with primary and secondary antibodies the membrane is placed in the appropriate substrate solution Protein bands usually appear within a few minutes Materials Membrane with transferred proteins and probed with antibody enzyme complex see Basic Protocol 2 or Alternate Protocol 3 TBS 4PPENDIX 24 Chromogenic visualization solution Table 6 2 1 Additional reagents and equipment for gel photography 1 If final membrane wash see Basic Protocol 2 step 7 or see Alternate Protocol 3 step 8 was performed in TTBS wash membrane 15 min at room temperature in 50 ml TBS The Tween 20 in TTBS interferes with 4CN development Bjerrum et al 1988 2 Place membrane into chromogenic visualization solution Bands should appear in 10 to 30 min 3 Terminate reaction by washing membrane in distilled water Air dry and photograph for a permanent record Current Protocols in Cell Biology BASIC PROTOCOL 3 Electrophoresis and Immunoblotting 6 2 11 Table 6 2 1 Chromogenic and Luminescent Visualization Systems System Reagent Reaction Detection Comments Chromogenic HRPO based 4CN DAB NiCl TMB AP based BCIP NBT Luminescent HRPO based Luminol H 0 p iodophenol AP based Substituted 1 2 dioxetane phosphates e g AMP
376. sfer the gel to a clean plastic container having 10 gel vol deionized water and incubate exactly 20 sec 11 Change the water in the container and incubate again exactly 20 sec The plastic container used in steps 10 and 11 can be reused in steps 14 and 15 but should first be rinsed with deionized water Develop the gel stain 12 Transfer the gel to a clean plastic container having 10 gel vol developer solution 13 Incubate with gentle agitation until the protein band s of interest becomes visible or until the gel matrix begins to get too dark This step lasts a few minutes S10 min and requires continuous visual inspection as the protein bands may develop rapidly and long incubations may result in high background staining Since some developing will still occur during steps 14 and 15 it is advisable to proceed to the next step as soon as the staining is judged to be optimal 14 Transfer the gel to a clean plastic container having 10 gel vol deionized water Incubate 30 sec 15 Change the water in the container and incubate an additional 30 sec 16 Transfer the gel to a clean plastic container having 10 gel vol 50 methanol 12 acetic acid in deionized water Incubate 10 min with gentle agitation 17 Remove the solution from the container and add 10 gel vol of 50 methanol The gel can be stored in this solution for several months at 4 C Alternatively the gel can be rehydrated by soaking in water for 5 to 10 min soak
377. should be used near the critical micelle concentration CMC 0 001 to 1 depending on the detergent The gel concentration has a dramatic effect on resolution and should be optimized in order to achieve the best separation and band sharp ness In general increasing the T will im prove band sharpness Troubleshooting Gel polymerization at acid pH can be prob lematic and sodium sulfite is needed for effi cient polymerization Andrews 1986 Both the ammonium persulfate and the sodium sul fite must be freshly made and the highest quality reagents available should be used Fur thermore the gel solutions should be at room temperature for effective polymerization If the protein does not enter the gel and no stained material is present at the well surface try reversing the polarity of the electrode If material concentrates at the top of the gel try lowering the acrylamide concentration Stained material at the top of the gel may also indicate poor solubilization and increasing the ionic strength of the solubilization buffer or adding a small amount of urea and or nonionic deter gent may be required Anticipated Results Proteins will resolve depending on their solubility and native charge at the chosen pH Ideally a distinct band representing the protein of interest will be visible If the band is diffuse then increasing the gel concentration or using a gradient gel will improve resolution If the band is not visible t
378. silver bromide crystals activated by the radioactivity or the light emitted from the screen Table 6 3 1 Different Methods for Isotope Detection and Their Sensitivities Enhancement Isotope Method Sensitivity over direct autoradiography 1257 S 100 16 32p S 50 10 5 14C F 400 15 PS F 400 15 3H F 8000 gt 100 Exposures conducted at 70 C using preexposed film b S intensifying screen F fluorography using PPO Defined as dpm cm2 required for detectable image A549 0 02 in 24 hr dDirect autoradiography for comparison was performed on Kodirex film Laskey 1980 Current Protocols in Cell Biology SUPPORT PROTOCOL 2 Electrophoresis and Immunoblotting 6 3 5 SUPPORT PROTOCOL 3 Detection and Quantitation of Radiolabeled Proteins in Gels and Blots 6 3 6 PREFLASHING PREEXPOSING FILM Silver bromide crystals that are activated by light particles or y rays are highly unstable and quickly revert back to their stable form The absorption of several photons increases their stability but does not ensure development approximately five photons of light are required to obtain a 50 probability that any single silver bromide crystal will be developed during film processing This inefficiency means that film images produced by very low levels of exposure will be disproportionately faint However two measures can be taken to maximize efficiency and linearity of exposure at the low levels c
379. so that it is firmly in contact with the stacking gel of the second dimension gel Remove excess running buffer 11 Overlay the IPG gel strip with the agarose equilibration buffer from step 6 and allow agarose to solidify 12 Carefully pour electrophoresis buffer into the upper reservoir taking care to avoid disturbing the agarose embedded IPG DryStrip 13 Connect electrodes and run the gels See UNIT 6 1 for electrophoresis conditions PREPARING MOLECULAR WEIGHT STANDARDS FOR TWO DIMENSIONAL GELS Molecular weight markers are usually necessary for the identification of proteins or as references to describe experimental proteins on two dimensional gels In many cases molecular weight markers are required only at the beginning of a project Once the system is established common proteins in the sample e g actin or tubulin provide sufficient references for molecular weight identification on subsequent gels To minimize any differences in migration of the molecular weight standards and isoelectric focused proteins the standard proteins should be loaded on the second dimension gel in the same manner as the IEF gel This protocol describes the preparation of standards in solidified agarose The agarose pieces may be stored at 80 C for at least a year and provide a convenient source of standards for the second dimension gel The procedure described is recommended for 3 mm IEF gels Narrow standards in solidified agarose made in tubes
380. specificity between the Pro Q glyco protein detection kit with concanavalin A the Pro Q glycoprotein detection kit with wheat germ agglutinin and the Pro Q Emerald 300 glycoprotein stain kits can be exploited in de fining structural features of glycans on glyco proteins Time Considerations The time considerations and number of steps required to detect glycoproteins using the Pro Q Emerald 300 Glycoprotein Gel Stain Kit Pro Q Emerald 300 Glycoprotein Blot Stain Kit and Pro Q Glycoprotein Detection Kit with Concanavalin A are summarized in Table 6 8 1 and contrasted with alternative glycoprotein detection technologies The methods can be completed in 2 to 4 hr and require 5 to 7 steps to complete This compares favorably with other methods that may require as much as 6 hr and 11 steps to complete Literature Cited Beeley J 1985 Glycoproteins and proteoglycan techniques In Laboratory Techniques in Bio chemistry and Molecular Biology R Burdon and P van Knippenberg eds vol 16 pp 5 28 Elsevier Press New York Hirabayashi J Arata Y and Kasai K 2001 Gly come project Concept strategy and preliminary application to Caenorhabditis elegans Pro teomics 1 285 294 Koch G and Smith M 1990 The analysis of glycoproteins in cells and tissues by two dimen sional polyacrylamide gel electrophoresis Elec trophoresis 11 213 219 Koketsu M and Linhardt R 2000 Electrophoresis for the analysis of acidic o
381. standards are commercially available as prepared mixtures see Table 6 1 3 Pour the stacking gel 7 10 11 Pour off the layer of H20 saturated isobutyl alcohol and rinse with 1x Tris Cl SDS pH 8 8 Residual isobutyl alcohol can reduce resolution of the protein bands therefore it must be completely removed The isobutyl alcohol overlay should not be left on the gel longer than 2 hr Prepare the stacking gel solution as directed in Table 6 1 1 Use the solution immediately to keep it from polymerizing in the flask Using a Pasteur pipet allow the stacking gel solution to trickle slowly into the center of the sandwich along an edge of one of the spacers until the height of the solution in the sandwich is 1 cm from the top of the plates Be careful not to introduce air bubbles into the stacking gel Insert a 0 75 mm Teflon comb into the layer of stacking gel solution If necessary add additional stacking gel to fill the spaces in the comb completely Again be careful not to trap air bubbles in the tooth edges of the comb they will cause small circular depressions in the well after polymerization that will lead to distortion in the protein bands during separation Allow the stacking gel solution to polymerize 30 to 45 min at room temperature A sharp optical discontinuity will be visible around the wells on polymerization Again failure to form a firm gel usually indicates a problem with the ammonium persulfate
382. ster first followed by progressively heavier solution 3 Prepare light and heavy acrylamide gel solutions Table 6 1 10 Use 12 ml of each solution for five 0 75 mm thick minigels Adjust volumes if a different thickness or number of gels is needed Do not add ammonium persulfate until just before use Deaeration is not recommended for gradient gels 4 With the outlet and interconnecting valve closed add the heavy solution to the reservoir chamber Briefly open the interconnecting valve to let a small amount of heavy solution through to the mixing chamber clearing the valve of air 5 Fill the mixing chamber with light solution Add 4 ul TEMED per 12 ml acrylamide solution to each chamber and mix with a disposable pipet Form the gradient and cast the gels 6 Turn on the magnetic stirrer Open the interconnecting valve and allow the chambers to equilibrate Then slowly open the outlet port to allow the solution to flow from the gradient maker to the multiple caster by gravity a peristaltic pump may be used for better control Adjust the flow rate to 3 to 4 ml min Faster flow rates are possible and will also produce good gradients However a fast flow increases the potential for introduction of bubbles into the caster 7 Close the outlet port as the last of the gradient solution leaves the mixing chamber just before air enters the outlet tube Fill the two chambers with plug solution and slowly open the outlet once again 8 A
383. sualize the protein bands and the careful disposal of this potentially toxic dye see Basic Protocol 3 Documentation Gels stained with Coomassie blue silver or zinc can be stored at 4 C for several weeks without significant decrease in sensitivity For long term storage gels stained with Coomassie blue silver or SYPRO Ruby can be equili brated in 2 w v glycerol and dried on a gel dryer although in the case of SYPRO Ruby stained gels this can result in decreased sensi tivity For photography or digital image acqui sition the blue bands of Coomassie blue stained gels have less contrast than the dark brown black bands of silver stained gels and SYPRO Ruby stained gels require the use of an appropriate photographic film e g Polar oid 667 black and white film or a sensitive CCD camera Negatively zinc stained gels can Electrophoresis and Immunoblotting 6 6 9 Supplement 6 Staining Proteins in Gels 6 6 10 Supplement 6 be problematic for photography or imaging on a CCD camera Quantitative analysis Scanning densitometric analyses of protein bands from stained polyacrylamide gels have been used to determine the relative abundance of proteins in complex mixtures the purity of protein samples and even the stoichiometry of multisubunit protein complexes These quanti tative analyses are performed assuming identi cal staining properties of the different proteins in the sample i e that the
384. t 80 C until ready to run the second dimension gel Gels may be stored at least 2 to 3 months at 80 C Do not place in the equilibration buffers required for the second dimension prior to storage ELECTROPHORESIS ON IMMOBILIZED pH GRADIENT GELS In this protocol after precast IEF gels Immobiline DryPlates from Amersham Pharma cia Biotech are rehydrated samples are loaded and subjected to isoelectric focusing Gels are typically run at 2500 to 3500 V and require focusing times of 2 to 7 hr Protein samples may be detected by conventional methods such as Coomassie blue or silver staining Isoelectric points can be determined with the use of pI calibration proteins alternatively because the gradient is linear one can measure the migration distance across the gel and estimate the pI at each location As noted in Basic Protocol 2 Immobiline DryPlates can be cut into 3 mm wide strips to use as the first dimension of two dimensional gels since Current Protocols in Cell Biology SUPPORT PROTOCOL 2 Electrophoresis and Immunoblotting 6 4 13 Supplement 4 Two Dimensional Gel Electrophoresis 6 4 14 Supplement 4 DryPlates are available in narrower pH ranges than DryStrips DryPlates can also be used as described in this protocol to simultaneously separate multiple samples in a single dimension Applications of this method include initial screening of samples to determine the optimal pH gradient prior to runnin
385. t so do not leave the filter in this solution gt 15 min Pour off excess guani dine HCl and then rinse the membrane several times in 1x TTBS Reblock the membrane and proceed with the standard immunoblotting pro cedure Membranes stripped using this proce dure can generally be reused three or four times Troubleshooting There are several problems associated with immunoblotting The antigen is solubilized and electrophoresed in the presence of denaturing agents e g SDS or urea and some antibodies may not recognize the denatured form of the antigen transferred to the membrane The re sults observed may be entirely dependent on the denaturation and transfer system used For example zwitterionic detergents have been shown to restore the antigenicity of outer mem brane proteins in immunoblotting Mandrell and Zollinger 1984 Gel electrophoresis under nondenaturing conditions can also be per formed Other potential problems include high back ground nonspecific or weak cross reactivity of antibodies poor protein transfer or membrane binding efficiency and insufficient sensitivity For an extensive survey and discussion of im munoblotting problems and artifacts see Bjer rum et al 1988 If no transfer of protein has occurred check the power supply and electroblot apparatus to make sure that the proper electrical connections were made and that power was delivered during transfer In addition check that the correct ori
386. t in a 4 solution Adjust volumes as necessary Marker mix 1 20 ul 1 M bis Tris stock solution pH adjusted to 7 0 with HCl 20 ul 1 M NaCl 143 ul 70 v v glycerol 5 mg ferritin 440 kDa and 880 kDa 5 mg catalase 232 kDa 5 mg bovine serum albumin 66 kDa and 132 kDa H20 to 1 ml Store at 4 C stable at least 1 year Marker mix 2 20 ul 1 M bis Tris stock solution pH adjusted to 7 0 with HCl 20 ul 1 M NaCl 143 ul 70 v v glycerol 5 mg thyroglobulin 670 kDa 5 mg aldolase 158 kDa H20 to 1 ml Store at 4 C stable at least 1 year Native BN transfer buffer 5 81 g Tris base 48 mM final 2 93 g glycine 39 mM final 200 ml methanol 20 final H20 to 1 liter Store at 4 C stable at least 1 year Pervanadate 100x Mix in the following order 50 ul 50 mM sodium orthovanadate 57 ul H2O 15 ul 30 H20 Incubate 5 to 30 min at room temperature becomes brownish Prepare fresh Current Protocols in Cell Biology Phosphate buffered saline PBS 10x 152 g NaCl 130 mM final 24 g monobasic sodium phosphate anhydrous 10 mM final 1600 ml H2O Adjust pH to 7 4 with NaOH Add H20 to 2 liters Store at room temperature stable at least 1 year PBS with 1 w v SDS 100 ml of 10x PBS see recipe 1 x final 100 ml 10 w v SDS stock solution APPENDIX 2A 1 final Add H20 to 1 liter Store at room temperature stable at least 1 year Protease and phosphatase inhibitor stock solutions
387. tain solution into a small clean plastic dish For one or two standard size mini blots use 50 ml to 100 ml of staining solution for larger blots use 500 to 750 ml 15 Place the air dried blot face down onto the surface of the staining solution and gently agitate e g on an orbital shaker at 50 rpm for 15 min at room temperature 16 After staining rinse the blot in four changes of water for 1 min each to decrease background fluorescence 17 Allow blots to air dry and visualize the stain using an appropriate method The stained blot is best viewed on a standard 300 nm UV epi illuminator though stain will be visible using a 254 nm UV C or 365 nm UV A epi illuminator Blots may also be visualized using various laser scanners 473 nm SHG laser 488 nm argon ion laser or 532 nm YAG laser Alternatively use a xenon arc lamp blue fluorescent light or blue light emitting diode LED source Blots may be photographed by Polaroid or CCD camera Use Polaroid 667 black and white print film and the SYPRO protein gel stain photographic filter Molecular Probes Exposure times vary with the intensity of the illumination source for an f stop of 4 5 1 to 3 sec should be required FLUORESCENT DETECTION OF GLYCOPROTEINS CONTAINING TERMINAL o MANNOPYRANOSYL AND o GLUCOPYRANOSYL RESIDUES ON ELECTROBLOT MEMBRANES Lectins are sugar binding proteins of nonimmune origin capable of agglutinating cells or precipitating glycoconjugates Beeley
388. tal amount of destaining solution and the destaining time can be reduced by placing a piece of adsorbent material with affinity for the Coomassie dye e g Whatman 3MM filter paper inside the container having the gel in destaining solution NOTE Avoid excessive incubation of the gel with destaining solution as it can result in decreased sensitivity due to dissociation of protein dye complexes 7 Add 10 gel vol storage solution and incubate for 10 to 15 min with gentle agitation At this point the gel can be stored at 4 C for several months in a plastic container or sealed plastic bag containing storage solution Alternatively it can be soaked in 2 v v glycerol for 15 to 30 min placed onto Whatman 3MM filter paper and subsequently dried on a vacuum system STAINING PROTEIN GELS WITH COOMASSIE BLUE AFTER ISOELECTRIC FOCUSING In polyacrylamide gel isoelectric focusing proteins are resolved based on their isoelectric points on a pH gradient generated by a mixture of ampholytes Many commercially available ampholytes bind Coomassie blue and therefore interfere with protein detection To overcome this problem the following procedure involves treatment with trichlo roacetic acid TCA to fix proteins while removing ampholytes and uses CuSO in the staining solution to help reduce the background Righetti and Drysdale 1974 Additional Materials see also Basic Protocol 1 Polyacrylamide isoelectric focusing gel with protein s of int
389. te and TEMED Swirl gently to mix Use immediately ADDITIONAL REAGENTS USED IN GELS 4x Tris Cl pH 6 8 0 5 M Tris Cl Dissolve 6 05 g Tris base in 40 ml H20 Adjust to pH 6 8 with 1 N HCl Add H20 to 100 ml total volume Filter the solution through a 0 45 1m filter and store up to 1 month at 4 C 8x Tris Cl pH 8 8 3 0 M Tris Cl Dissolve 182 g Tris base in 300 ml H20 Adjust to pH 8 8 with 1 N HCI Add H20 to 500 ml total volume Filter the solution through a 0 45 um filter and store up to 1 month at 4 C The recipes produce 15 ml of separating gel and 5 ml of stacking gel which are adequate for one gel of dimensions 0 75 mm x 14 cm x 14 cm The recipes are based on the modified Laemmli peptide separation system of Okajima et al 1993 Pall reagents and solutions used in the protocol must be prepared with Milli Q purified water or equivalent Best to prepare fresh Failure to form a firm gel usually indicates a problem with the ammonium persulfate TEMED or both Current Protocols in Cell Biology Electrophoresis and Immunoblotting 6 1 13 Supplement 37 3 Run the gel see Basic Protocol 1 steps 21 and 22 Note that the separations will take 25 longer than those using Basic Protocol 1 The increased buffer concentrations lead to faster transit through the stacking gel but lower mobility in the resolving gel 4 Disassemble the gel see Basic Protocol 1 steps 23
390. ted by Esteban C Dell Angelica and Juan S Bonifacino Current Protocols in Cell Biology 2000 6 6 1 6 6 14 Copyright 2000 by John Wiley amp Sons Inc UNIT 6 6 BASIC PROTOCOL 1 Electrophoresis and Immunoblotting 6 6 1 Supplement 6 ALTERNATE PROTOCOL 1 Staining Proteins in Gels 6 6 2 Supplement 6 NOTE All steps should be performed at room temperature 1 Remove polyacrylamide gel from electrophoresis assembly and place it in a plastic container with lid containing a large excess 10 gel vol of Coomassie blue staining solution 2 Incubate with gentle agitation for 220 min for a gel lt 1 mm thick or 21 hr for a gel gt 1 mm thick The use of a platform shaker is recommended Most proteins are fixed due to the presence of methanol in the staining solution gels can therefore be left in staining solution for many hours i e overnight without any adverse effect unless the protein s of interest is very small lt 5 kDa and can be lost by diffusion due to incomplete fixation 3 Remove the Coomassie blue staining solution and rinse the gel briefly with distilled water The Coomassie blue staining solution can be reused several times 4 Add 10 gel vol of destaining solution and incubate with gentle agitation until the solution becomes as dark as the gel matrix 5 Discard the destaining solution 6 Repeat steps 4 and 5 several times until a clear background is obtained Both the to
391. ted levels of image enhancement obtained through fluorography are listed in Table 6 3 1 Sodium salicylate can also be used for fluorography as described below Chamberlain 1979 It yields levels of image enhancement comparable to organic scintillants although it sometimes causes a more diffuse film image The conditions should work for most standard sizes and thicknesses of gels CAUTION Gloves should be worn at all times sodium salicylate can elicit allergic reactions and is readily absorbed through the skin Materials Polyacrylamide gel 1 M sodium salicylate pH 5 to 7 freshly prepared Additional reagents and equipment for fixing and drying gels see Support Protocol 1 1 If gel is acid fixed soak for 1 to 5 hr in 20 vol water to prevent precipitation of salicylic acid from the sodium salicylate 2 Soak gel 30 min in 10 vol of 1 M sodium salicylate pH 5 to 7 To prevent cracking of gels with gt 15 acrylamide or thicker than 1 5 mm 2 v v glycerol can be added to the 1 M sodium salicylate 3 Dry the gel see Support Protocol 1 and proceed with autoradiography see Basic Protocol DENSITOMETRY Film images obtained by autoradiographic methods can be quantified by densitometry Densitometers work by comparing the intensity of light transmitted through a sample with the intensity of the incident light The amount of light transmitted will be proportional to the amount of radioactivity in the gel provided that the
392. tein is laid on a sheet of filter paper The uncovered side of the gel is overlaid with a sheet of membrane precut to the size of the gel plus 1 to 2 mm on each edge then this membrane is overlaid with another sheet of filter paper The filter paper containing the gel and membrane is sandwiched between Scotch Brite pads This sandwich is placed in a plastic support and the entire assembly is placed in a tank containing transfer buffer For transfer of negatively charged protein the membrane is positioned on the anode side of the gel For transfer of positively charged protein the membrane is placed on the cathode side of the gel Charged proteins are transferred electrophoreti cally from the gel onto the membrane Transfer is achieved by applying a voltage of 100 V for 1 to 2 hr with cooling or 14 V overnight Current Protocols in Cell Biology 5 Place gel on top of filter paper The side of the gel touching the paper arbitrarily becomes the cathode side of the gel i e ultimately toward the negative electrode when positioned in the tank Remove any air bubbles between gel and filter paper by gently rolling a test tube or glass rod over surface of gel Any bubbles between the filter paper gel and membrane will block current flow and prevent protein transfer This problem is indicated on the membrane by sharply defined white areas devoid of transferred protein Proteins are usually negatively charged in transfer buffer and move toward the p
393. tein protein interactions Here we give detailed protocols for the separation of multiprotein complexes by two dimensional BN SDS PAGE and for a related technique to determine the stoichiometry of these complexes Curr Protoc Cell Biol 38 6 10 1 6 10 21 2008 by John Wiley amp Sons Inc Keywords multiprotein complex e native e gel electrophoresis e two dimensional e Coomassie blue e protein protein interaction INTRODUCTION Blue Native Polyacrylamide Gel Electrophoresis BN PAGE Basic Protocol 1 separates native proteins and protein complexes independently of their individual isoelectric points with high resolution capacity This is done by conferring a negative charge on all proteins via the dye Coomassie blue The separation depends mainly on the size of the protein complex In combination with a second dimension sodium dodecyl sulfate PAGE SDS PAGE in a perpendicular direction Basic Protocol 2 it is an excellent choice to identify and characterize multiprotein complexes It allows the determination of the size subunit composition and relative abundance of different multiprotein complexes from total cell and tissue homogenates as well as from purified material In addition it is used for a one step preparative purification of protein complexes One critical step in performing high quality BN PAGE is the preparation of the sample which has to be devoid of potassium and divalent cations The most useful source for protein
394. teins in the gel shown in Figure 6 1 9 were separated in a 5 1 to 20 5 T acrylamide gradient The T of the point halfway through the resolving gel is 12 5 T Simply plotting log molecular mass versus distance moved into the gel or R also pro duces a relatively linear standard curve over a fairly wide size range If two proteins have identical molecular sizes they more than likely will not be resolved with one dimensional SDS PAGE and two dimensional SDS PAGE should be consid ered Unusual protein compositions can cause anomalous mobilities during electrophoresis see Critical Parameters and Troubleshoot ing but similar sized proteins of widely dif ferent amino acid composition or structure may still be resolved from one another using one dimensional SDS PAGE Purified protein complexes or multimeric proteins consisting of subunits of different molecular size will be resolved into constituent polypeptides Com parison of the protein bands obtained under nonreducing and reducing conditions provides information about the molecular size of disul fide cross linked component subunits The in dividual polypeptides can be isolated by elec troelution or electroblotting and the amino acid sequences can be determined Both the tricine Schagger and von Jagow 1987 and the modified Laemmli Okajima et al 1993 peptide separation procedures presented here Alternate Protocols 1 and 2 are simple to set up and provide resolution d
395. ter paper stack Remove all bubbles between membrane and filter paper by rolling a test tube over surface of membrane Any bubbles in the filter paper stack or between the filter paper membrane and gel will block current flow and prevent protein transfer This problem is indicated on the membrane by sharply defined white areas devoid of transferred protein Current Protocols in Cell Biology Electrophoresis and Immunoblotting 6 2 5 ALTERNATE PROTOCOL 2 Immunoblotting and Immunodetection 6 2 6 6 Place gel on top of membrane Gently roll a test tube over surface of gel to ensure intimate contact between gel and membrane and to remove any interfering bubbles Poor contact between the gel and membrane will cause a swirled pattern of transferred proteins on the membrane Some proteins will transfer as soon as the gel is placed on the membrane repositioning the gel or membrane can result in a smeared or double image on the developed blot 7 Complete the transfer stack by putting the three remaining sheets of filter paper on top of gel Roll out bubbles as described above Multiple gels can be transferred using semidry blotting Simply put a sheet of porous cellophane Amersham Pharmacia Biotech or dialysis membrane Bio Rad or Sartorius equilibrated with transfer buffer between each transfer stack Fig 6 2 2 Transfer effi ciency is dependent on the position of the transfer stack in the blotting unit and for critica
396. ternate Protocol and were 0 2 n electrophoresed 16 hr at 6 mA Proteins were stained with Coomassie blue 0 3 5 as 0 6 0 7 0 8 0 9 1 0 A B 1 0 oa D e Bt pa 0 10 Patty rar Poa ale det PSS ee eye ee epi 4 6 8 10 12 14 4 6 8 10 12 14 T T Figure 6 5 2 Effect of T on the relative mobility of several native proteins The relative mobility R of the standard proteins shown in Figure 6 5 1 was determined at four different gel concentra tions and plotted as log Ry against T See text for details A BSA monomer squares and dimer circles B carbonic anhydrase isoforms Current Protocols in Cell Biology Electrophoresis and Immunoblotting 6 5 7 Supplement 5 One Dimensional Electrophoresis Using Nondenaturing Conditions 6 5 8 Supplement 5 0 3 Log K oO S O 0 01 l 14 29 45 66 132 545 Log molecular weight Figure 6 5 3 Native molecular weight standard curve The log slope of the line K from Figure 6 5 2 is plotted against log molecular weight of the standards REAGENTS AND SOLUTIONS Use Milli O purified water or equivalent for the preparation of all buffers For common stock solutions see APPENDIX 24 for suppliers see SUPPLIERS APPENDIX Acetic acid gel buffer 4x 200 mM acetic acid pH 3 7 to 5 6 11 49 ml glacial acetic acid Add to 500 ml H O Adjust to pH 3 7 to 5 6 with 1 M NaOH Add H O to 1000 ml Store u
397. the top of the well arm If the protein bands are uneven the stack ing gel may not have been adequately poly merized This can be corrected by deaerat ing the stacking gel solution thoroughly or by increasing the ammonium persulfate and TEMED concentrations by one third to one half Another cause of distorted bands is salt in the protein sample which can be removed by dialysis gel filtration or precipitation Skewed protein bands can be caused by an uneven in terface between the stacking and separating gels which can be corrected by starting over and being careful not to disturb the separating gel while overlaying with isobutyl alcohol If a run takes too long the buffers may be too concentrated or the operating current too low If the run is too short the buffers may be too dilute or the operating current too high If double bands are observed the protein may be partially oxidized or partially de graded Oxidation can be minimized by in creasing the 2 ME concentration in the sample buffer or by preparing a fresh protein sample If fewer bands than expected are observed and there is a heavy protein band at the dye front increase the acrylamide percentage in the gel Anticipated Results Polyacrylamide gel electrophoresis done under denaturing and reducing conditions should resolve any two proteins except two of identical size Resolution of proteins in the presence of SDS is a function of gel con centration and the size
398. the gels Do not substantially exceed 16 hr as extensive incubation especially rehydrating gels over a weekend increases potential problems due to evaporation and subsequent urea crystallization In addition long incubation times increase the extent of urea decomposition which will increase the risk of amino group modification on proteins by the cyanate produced from urea decomposition Electrophoresis Ifthe IPGphor system is used a low voltage 30 to 40 V can be applied during rehydration and which improves isoelectric focusing and protein yields of some samples With this device Immunoblotting 6 4 11 Current Protocols in Cell Biology Supplement 4 Two Dimensional Gel Electrophoresis 6 4 12 Supplement 4 6 protocols can be preprogrammed so as to allow isoelectric focusing to immediately follow rehydration Hence both rehydration with low voltage and isoelectric focusing can be completed overnight since 8 hr is sufficient for rehydration and up to 8000 V can be used in later stages of isoelectric focusing to shorten the total focusing time After the overnight rehydration slide the lid off the reswelling tray Place a forceps tip into the slight depression under each strip and remove the strip Gently blot any excess oil or moisture from the plastic backing of the rehydrated strips with filter paper A damp piece of filter paper may also be used to blot the surface of the gel Some of the paper may adhere to the
399. the plastic wrap and lay the blot face down onto the solution being careful not to trap air bubbles The time required for optimal staining must be determined empirically because the substrate turnover rate depends on the amount of glycoprotein on the blot Generally a 5 to 20 min incubation is sufficient but overnight incubation is permissible Do not wash the blot after staining as this will cause extensive loss of signal The blot may be air dried however Visualize the fluorescent DDAO product using either UV epi illumination and a digital or film camera or using a laser equipped with a 633 nm helium neon laser or 635 nm diode laser source For UV epi illumination place the blot signal side up on a flat surface For highest sensitivity and lowest background use a UV blocking filter such as the SYPRO gel stain photographic filter Long pass filters with a cutoff at 630 nm are ideal for CCD cameras For laser scanners place the blot signal side down on the scanner bed For highest sensitivity match the light sources and filters of the instrument as closely as possible to the absorbance maximum 646 nm and emission maximum 659 nm of DDAO If desired the Con A AP complex can be stripped off of the blot and the blot reprobed with another lectin AP complex or an antibody AP complex To strip incubate the blot in stripping buffer for 40 min at 50 C with gentle agitation Then wash the blot in wash buffer two times for 5 min each at
400. the voltage and current source Most power supplies deliver constant current or constant voltage Some will also deliver constant power power voltage x current or VI IR The discussion below focuses on constant current because this is the most common mode in vertical SDS PAGE Current Protocols in Cell Biology Most modern commercial equipment is color coded so that the red or positive terminal of the power supply can simply be connected to the red lead of the gel apparatus which goes to the lower buffer chamber The black lead is connected to the black or negative terminal and goes to the upper buffer chamber This configuration is designed to work with vertical slab gel electrophoreses in which negatively charged proteins or nucleic acids move to the positive electrode in the lower buffer chamber an anionic system When a single gel is attached to a power supply the negative charges flow from the negative cathode black terminal into the upper buffer chamber through the gel and into the lower buffer chamber The lower buffer chamber is connected to the positive anode red terminal to complete the circuit Thus negatively charged molecules such as SDS coated proteins and nucleic acids move from the negative cathode attached to the upper buffer chamber toward the positive anode attached to the lower chamber SDS PAGE is an anionic system because of the negatively charged SDS Occasionally proteins are separated in cationic systems I
401. ther applications for Ponceau S calibration include monitoring transfer effi ciency under varied conditions for optimization of tank and semidry blotting Immunoblotted proteins can be detected by chromogenic or luminescent assays see Table 6 2 1 for a description of the reagents available for each system their reactions and a compari son of their advantages and disadvantages Luminescent detection methods offer several advantages over traditional chromogenic pro cedures In general luminescent substrates in crease the sensitivity of both HRPO and phos phatase systems without the need for radioiso topes Substrates for the latter have only recently been applied to protein blotting see Gillespie and Hudspeth 1991 Sandhu et al 1991 Bronstein et al 1992 Luminescent de tection can be completed in as little as a few seconds exposures rarely go more than hr Depending on the system the luminescence can last for 3 days permitting multiple expo sures of the same blot Furthermore the signal is detected by film and varying the exposure can result in more or less sensitivity Lumines cent blots can be easily erased and reprobed because the reaction products are soluble and do not deposit on the membrane see below Compared to chromogenic development the luminescent image recorded on film is easier to photograph and to quantitate by densitometry Alkaline phosphatase based luminescent protocols that achieve maximum
402. thod in molecular biology for separat ing biomolecules This prominence is the result of several factors including the robustness speed and potentially high throughput of the technique The results of this method are tradi tionally documented using silver halide based photography followed by manual interpreta tion While this remains an excellent method for qualitative documentation of single gel re sults digital capture offers a number of signifi cant advantages when documentation requires quantitation and sophisticated analysis Digital images of gel electropherograms can be ob tained rapidly using an image capture device and the images can be easily manipulated using image analysis software REASONS FOR DIGITAL DOCUMENTATION AND ANALYSIS There are several reasons to consider digital documentation and analysis of electrophoresis results These justifications can usually be cate gorized into issues of ease of handling accu racy reproducibility and cost Ease of Handling A major advantage of the digital revolution has been in storage and retrieval of information Storage in notebooks and filing cabinets pre viously meant that searching for specific data or experiments was a tedious manual process With digital information modern search en gines can quickly find specific information in a fraction of the time usually required for a manual search Making backup copies of non digital data can be difficult expensive an
403. tical gel unit Hoefer Mighty Small SE 250 280 or Bio Rad Mini Protean II with glass plates clamps and buffer chambers 0 75 mm spacers Multiple gel caster Hoefer SE 275 295 or Bio Rad Mini Protean II multicasting chamber Acrylic plate Hoefer SE 217 or Bio Rad 165 1957 or polycarbonate separation sheet Hoefer SE 213 or Bio Rad 165 1958 10 and 50 ml syringes Combs Teflon Hoefer SE 211A series or Bio Rad Mini Protean II Long razor blade Micropipet Additional reagents and equipment for standard denaturing SDS PAGE see Basic Protocol 1 Pour the separating gel 1 Assemble each gel sandwich by stacking in order the notched Hoefer or small rectangular Bio Rad plate 0 75 mm spacers and the larger rectangular plate Be sure to align the spacers properly with the ends flush with the top and bottom edge of the two plates when positioning the sandwiches in the multiple gel caster Fig 6 1 4 The protocol described is basically for the Hoefer system For other systems make adjustments according to the manufacturers instructions Alternatively precast minigels can be purchased from a number of suppliers see Table 6 1 4 Current Protocols in Cell Biology faceplate inlet fitting bottom 23 X CE high acrylamide Se concentration lt i gel sandwich filler glass plate caster body concentration T shaped spacer Figure 6 1 4 Minigel sandwiches positioned in the multiple gel cast
404. ting with an atlas of immunoblotting artifacts Jn CRC Handbook of Immunoblotting of Proteins Vol I Technical Descriptions O J Bjerrum and N H H Heegaard eds pp 227 254 CRC Press Boca Raton Fla Blake M S Johnston K H Russell Jones G J and Gotschlich E C 1984 A rapid sensitive method for detection of alkaline phosphatase conjugated anti antibody on Western blots Anal Biochem 136 175 179 Bronstein I Voyta J C Murphy O J Bresnick L and Kricka L J 1992 Improved chemilumi nescent Western blotting procedure BioTech niques 12 748 753 Burnette W N 1981 Western blotting Electro phoretic transfer of proteins from sodium do decyl sulfate polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A Anal Biochem 112 195 203 Craig W Y Poulin S E Collins M F Ledue T B and Ritchie R F 1993 Background staining in immunoblot assays Reduction of signal caused by cross reactivity with blocking agents J Im munol Methods 158 67 76 Dionisi H M Checa S K and Viale A M 1995 Protein immunoblotting of stained gels BioTechniques 19 348 350 Gillespie P G and Hudspeth A J 1991 Chemilu minescence detection of proteins from single cells Proc Natl Acad Sci U S A 88 2563 2567 Harlow E and Lane D 1988 Immunoblotting In Antibodies A Laboratory Manual pp 471 510 CSH Laboratory Cold Spring
405. tinylated HRPO or AP and biotinylated secondary antibody request membrane immunodetection protocols when ordering 1 Equilibrate membrane in appropriate blocking buffer in heat sealed plastic bag with constant agitation using an orbital shaker or rocking platform For nitrocellulose and PVDF incubate 30 to 60 min at room temperature For nylon incubate 22 hr at 37 C TTBS is well suited for avidin biotin systems For nylon protein binding agents are recommended Because nonfat dry milk contains residual biotin which will interfere with the immunoassay it must be used in the blocking step only If membrane is to be stripped and reprobed see Support Protocol 3 blocking buffer must contain casein for AP systems or nonfat dry milk Plastic incubation trays are often used in place of heat sealable bags and can be especially useful when processing large numbers of strips in different primary antibody solutions 2 Prepare primary antibody solution in TTBS nitrocellulose or PVDF or TBS nylon Dilutions of sera containing primary antibody generally range from 1 100 to 1 100 000 This depends in large part on the sensitivity of the detection system With high sensitivity avidin biotin systems dilutions from 1 1000 to 1 100 000 are common Higher dilutions can be used with AP or luminescence based detection systems To determine the appro priate concentration of the primary antibody a dilution series is easily performed with membrane
406. tions of electrodes and electrode solutions reversed in the electro phoresis chamber The total protein load per gel depends on the complexity of the sample the solubility of pro teins in the sample and the diameter of the first dimension gel Approximately 4 times as much total sample can be applied to a 3 mm gel com pared with a 1 5 mm gel Another advantage of a larger diameter isoelectric focusing gel is that extrusion and gel handling are easier owing to improved strength of the gel If care is exercised in loading the first dimension gel onto the 1 5 mm second dimension gel the final resolution will be similar to that obtained using smaller di ameter IEF gels The separating or resolving power of a system is dependent on the quality of ampholytes used the slope of the pH gradient and the lengths of both first and second dimen sion gels Immobilized pH gradient gels In immobilized pH gradient IPG gels Basic Protocol 2 the pH gradient is an integral part of the polyacrylamide matrix Strahler and Hanash 1991 Because the pH gradient is covalently associated with the polyacrylamide gel matrix precise reproducible and very high resolution separations can be achieved A variety of precast gels and all the necessary equipment are commer cially available from either Amersham Pharmacia Biotech or Bio Rad Reproducible two dimen sional gels can be obtained by running a sample on a narrow strip of immobilized pH gradi
407. tipped needle attached to a syringe Attach gel sandwich to upper buffer chamber following manufacturer s instructions Fill lower buffer chamber with the recommended amount of 1 x SDS electrophoresis buffer Place sandwich attached to upper buffer chamber into lower buffer chamber Partially fill the upper buffer chamber with 1x SDS electrophoresis buffer so that the sample wells of the stacking gel are filled with buffer Monitor the upper buffer chamber for leaks and if necessary reassemble the unit A slow leak in the upper buffer chamber may cause arcing around the upper electrode and damage the upper buffer chamber Using a 25 or 100 ul syringe with a flat tipped needle load the protein sample s into one or more wells by carefully applying the sample as a thin layer at the bottom of the wells Load control wells with molecular weight standards Add an equal volume of 1x SDS sample buffer to any empty wells to prevent spreading of adjoining lanes Disposable loading tips can be used with automatic pipettors to simplify loading Preparing the samples at approximately the same concentration and loading an equal volume to each well will ensure that all lanes are the same width and that the proteins run evenly If unequal volumes of sample buffer are added to wells the lane with the larger volume will spread during electrophoresis and constrict the adjacent lanes causing distortions The samples will layer on the bottom of the
408. tly Remove comb slowly Use level to verify that the apparatus is level Decrease power settings Cool buffer to 4 C Circulate buffer Flush wells with electrophoresis buffer Inspect wells for trapped air bubbles Verify pH of buffer Circulate buffer during run Increase concentration and or time of blocking step Remove membrane from substrate after 1 min Wash or replace fiber pads and clean apparatus Increase wash time and volume Review recipe especially Tween 20 Review supplier s recommendations Run test strips to optimize reactions Increase amount of sample Increase antibody concentration Increase transfer time Stain membrane for protein transfer Mix more thoroughly Agarose Gel Electrophoresis of Proteins 6 7 10 Supplement 15 COMMENTARY Background Information The major use of agarose in protein analysis is its application as a matrix for molecular sieving however it is also widely used as a material that can be modified to form an affinity matrix for affinity chromatography Because of its ability to separate proteins of very large size it is also utilized for electrophoresis and prepa ration of very large proteins ranging from sev eral million to approximately fifty thousand daltons Agarose has the advantage of being nontoxic In addition it may be melted to allow recovery and further studies of the separated protein and an excised band may be directly injected into an an
409. tly transferred to membranes unir6 2 and detected using radiolabeled probes such as antibodies and 5J labeled protein A The autoradio graphic image whether generated on film or a phosphor screen reflects the distribution of the radioactive proteins on the two dimensional surface of the gel or filter Molecular sizes of radiolabeled proteins therefore can be deter mined by correlating their positions with mo lecular markers Also the density of the band images can be used to determine the relative Current Protocols in Cell Biology quantities of the radiolabeled proteins in the sample Critical Parameters The sensitivity of the detection device and the strength of the radioactive signal are the two most important parameters for autoradiogra phy Sensitivity can be enhanced by treating samples with fluors or by using intensifying screens Table 6 3 2 Because phosphor imag ing is 10 to 250 times more sensitive than film Johnston et al 1990 this technology makes it possible to monitor radioactive samples that would previously have gone undetected with film A second important parameter is the range over which the measurement device is linear Film requires preflashing in order for the inten sity of the image to be linear with respect to the amount of radioactivity particularly for weakly radioactive samples Laskey and Mills 1975 1977 Phosphor imaging offers a much wider linear range of measurement 5 orders of
410. tly to mix Use immediately STACKING GEL 3 9 w v acrylamide In a 25 ml side arm flask mix 0 65 ml of 30 acrylamide 0 8 bisacrylamide 1 25 ml of 4x Tris Cl SDS pH 6 8 see reagents below and 3 05 ml H20 Degas under vacuum 10 to 15 min Add 25 ul of 10 ammonium persulfate and 5 ul TEMED Swirl gently to mix Use immediately REAGENTS USED IN GELS 30 w v acrylamide 0 8 w v bisacrylamide Mix 30 0 g acrylamide and 0 8 g N N methylenebisacrylamide with H20 in a total volume of 100 ml Filter the solution through a 0 45 um filter and store at 4 C in the dark The 2x crystallized grades of acrylamide and bisacrylamide are recommended Discard after 30 days as acrylamide gradually hydrolyzes to acrylic acid and ammonia CAUTION Acrylamide monomer is neurotoxic A mask should be worn when weighing acrylamide powder Gloves should be worn while handling the solution and the solution should not be pipetted by mouth 4x Tris Cl SDS pH 6 8 0 5 M Tris Cl containing 0 4 w v SDS Dissolve 6 05 g Tris base in 40 ml H20 Adjust to pH 6 8 with 1 N HCI Add H20 to 100 ml total volume Filter the solution through a 0 45 um filter add 0 4 g SDS and store at 4 C up to month 4x Tris Cl SDS pH 8 8 1 5 M Tris Cl containing 0 4 w v SDS Dissolve 91 g Tris base in 300 ml H20 Adjust to pH 8 8 with 1 N HCl Add H20 to 500 ml total volume Filter the solution through a 0 45 um filter add 2 g SDS and store at 4 C up to month
411. to 26 Proteins in the gel may now be stained ALTERNATE CONTINUOUS SDS PAGE PROTOCOLS With continuous SDS PAGE the same buffer is used for both the gel and electrode solutions Although continuous gels lack the resolution of the discontinuous systems they are extremely versatile less prone to mobility artifacts and much easier to prepare The stacking gel is omitted Additional Materials also see Basic Protocol 1 Separating gel solution Table 6 1 8 2x and 1x phosphate SDS sample buffer see recipe 1x phosphate SDS electrophoresis buffer see recipe Table 6 1 8 Recipes for Separating Gels for Continuous SDS PAGE SEPARATING GEL Final acrylamide concentration in the separating gel Stock solution 5 6 7 8 9 10 11 12 13 14 15 30 w v 2 5 3 00 3 50 4 00 4 50 5 00 5 50 6 00 6 50 7 00 7 50 acrylamide 0 8 w v bisacrylamide 4x phosphate SDS 3 75 3 75 3 75 3 75 3 75 3 75 3 75 3 75 3 75 3 75 3 75 pH 7 2 H20 8 75 8 25 7 75 7 25 6 75 6 25 5 75 5 25 4 75 4 25 3 75 10 w v 0 05 0 05 0 05 0 05 0 05 0 05 0 05 0 05 0 05 0 05 0 05 ammonium persulfate TEMED 0 01 0 01 0 01 0 01 0 01 0 01 0 01 0 01 0 01 0 01 0 01 Preparation of separating gel In a 25 ml side arm flask mix 30 acrylamide 0 8 bisacrylamide solution see Table 6 1 1 4x phosphate SDS pH 7 2 and H20 Degas under vacuum about 5 min Add 10 ammonium persulfate and TEMED Swirl gently to mix Use immediately ADDITIONAL REAGENTS USED IN GELS 4x phosphate SDS pH 7 2 0 4 M s
412. to the above protocol includes ad dition of 0 3 w v CHAPS in the gel solution which is intended to improve the solubility of proteins and their migration and separation in the gel The system is supplied with a manual that adequately describes the method Critical Parameters and Troubleshooting Although no individual steps in two dimen sional gel analysis are exceptionally difficult the large number of steps involved increase the like lihood and possible severity of errors or problems Several steps are especially critical and may re quire optimization The first is sample prepara tion Proteins applied to IEF gels have to be completely solubilized Residual precipitate or even soluble aggregates are likely to cause arti facts on the end of the tube gel or position on the IPG strip where the sample is loaded Precipitated or aggregated proteins may also interact with soluble proteins causing components with nor mally good solubility to coprecipitate or migrate anomalously Two general rules of thumb apply to sample solubility 1 the more complex the sample or the more crude the extract the more likely problems will be encountered with sample solubility and 2 the higher the protein load applied to the gel the more likely solubility problems will arise Care must also be taken to avoid proteolysis during sample preparation especially when com plex impure samples are used If samples are frozen either before or after sol
413. tor based objects such as polygons and fonts It supports a lt 256 gray level LUT and monochrome images can be RLE compressed Because it only offers a 256 gray level LUT it has the same weaknesses that GIF does with true gray scale and high inten sity resolution images In addition any vector objects in the image are difficult to translate on a PC since they are designed to be interpreted by Macintosh QuickDraw routines BMPis the native bitmap file format present on Windows based PCs It supports 2 16 256 or 16 million level images With images of 256 gray levels itimplements a LUT while the highest resolution image is implemented directly RLE compression is optional for 16 and 256 gray level images Since compression is prohibited on 16 million gray level images and there is no intermediate level supported beyond 256 levels BMP is not a good choice for images with high intensity resolution re quirements ANALYSIS Once the image has been captured the data needs to be analyzed and distilled into informa tion about the results of the electrophoresis experiment Through the use of standards and experimenter input this software driven pro cess can estimate mass and quantity of objects in an image and detect relationships between objects within one image and between similar images The type of software used depends on the analysis to be performed Images from sin gle electrophonetic separations are examined by one di
414. tration of antibody run a preliminary gel followed by immunoblotting Cut the blot into several vertical test strips each containing I to 2 lanes Use the supplier s recom mended concentration of antibody as a starting point and process the test strips with 3 to 10 to 30 fold increased and decreased antibody concentrations in separate containers AGARAOSE GEL ELECTROPHORESIS WITH IN GEL ANTIBODY ANALYSIS This alternative protocol describes a method for separating large plasma proteins using SDS agarose electrophoresis and visualizing the protein of interest directly in the gel with a T radiolabeled rabbit antibody in this case to human von Willebrand factor protein and autoradiography see also uniT 6 3 The methodology applied to agarose electropho resis of von Willebrand factor is outlined below The gel preparation consists of mating two glass plates on a horizontal surface separated by a 0 5 mm spacer A sheet of GelBond support film is fixed to the glass plate to provide support for the agarose Agarose at a concentration of 1 35 w v is poured between the glass plate and the spacer plate After the gel is solidified the apparatus is disassembled and 1 0 x 0 1 cm wells are punched into the gel The gel is placed into a horizontal electrophoresis chamber presoaked wicks are attached to the gel and sample volumes of 8 ul are loaded The prepared samples are electrophoresed until the sample dye has migrated 8 to 10 cm The gel is i
415. trength of the samples loaded onto the gels An initial voltage of lt 800 V is recommended for 3 mm gels loaded with samples containing less than 100 mM salts buffers the voltage could be increased to 1200 V after 1 hr if cooling is used The current is a derivative of voltage and is never preset for isoelectric focusing purposes Some power supplies allow preprogramming the desired number of volt hours and continuously adjust voltage and current during the isoelectric focusing procedure constant power The total number of volt hours is a major factor that affects separation in the first dimension Optimal focusing time will vary for different ampholyte combina tions but 12 000 Vhr is a reasonable value for most systems To achieve a total of 12 000 Vhr set the power supply to 667 V for 18 hr These conditions are convenient for an overnight separation and do not require use of a cooling unit Higher voltages can be used but may cause overheating of gels unless a highly efficient cooling system is employed The maximum practical voltage decreases with increased gel tube inner diameter Focusing for too long may cause cathodic drift and result in a shifted pH profile in the gel whereas focusing for a short time will decrease resolution Extrude and store gels 27 Turn off power supply and carefully disconnect leads Detach the lid and remove the NaOH solution from the upper reservoir of the electrophoresis chamber using a 60 ml disposable plastic
416. troblot ting Towbin etal 1979 subsequently popu larized as western blotting or immunoblotting Burnette 1981 was conceived Immuno blotting is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen antibody recognition It involves the solubilization and electrophoretic separation of proteins glycoproteins or lipopolysaccharides by SDS PAGE unr 6 1 or urea PAGE fol lowed by quantitative transfer and irreversible binding to nitrocellulose PVDF or nylon This technique has been useful in identifying spe cific antigens recognized by polyclonal or monoclonal antibodies and is highly sensitive 1 ng of antigen can be detected Electroblotting of previously stained gels is a convenient way to visualize and document the gel prior to immunoblotting Transfer efficien cies at all molecular weights will be lower with fixed and stained gels This is particularly true of proteins gt 50 kDa Perides et al 1986 The additional incubation in 6 M urea will signifi cantly increase transfer efficiency of all pro teins and is required for proteins gt 50 kDa Ponceau S staining provides an easy method for calibrating and quantitating the amount of material on a nitrocellulose or PVDF blot An alternative to this method is to use an internal protein control with a separate antibody probe but these tend to be expensive and time con suming to use O
417. trols on how the light detectors report arange of light intensities Below is a brief description of each Contrast Contrast describes the slope of the light intensity response curve An increase in the contrast increases the slope of the curve The resultis amore detailed display over a narrowed range of intensities with less detail in the re maining portions of the intensity range This is depicted in Figure 6 9 1A and 6 9 1B where a normal unadjusted image and a contrast ad justed image are displayed respectively The contrast was increased on midrange intensity values in Figure 6 9 1B to highlight band inten sity differences at the expense of background information Images with a narrow range of informative intensities can benefit from in creasing the contrast since that effectively in creases the scale and improves detection of minor differences in intensity Contrast settings should be lowered if information is being lost outside of the contrast range For example in Figure 6 9 1B loss of background information between peaks indicates that this image should not be used for quantitation Brightness While brightness can have many different definitions only one will be considered here Brightness shifts the light intensity response curve without changing its slope as is shown in Figure 6 9 1C Another name for brightness is black level since itis commonly used to control the number of black picture elements pixels in a
418. tube Current Protocols in Cell Biology 13 14 15 16 17 18 19 20 Cap the tube to prevent evaporation vortex and transfer directly to a 100 C water bath for 3 to 5 min Let immunoprecipitates dissolve for 1 hr at 56 C in 1x SDS sample buffer prior to boiling DO NOT leave the sample in SDS sample buffer at room temperature without first heating to 100 C to inactivate proteases see Critical Parameters and Troubleshooting Endogenous proteases are very active in SDS sample buffer and will cause severe degradation of the sample proteins after even a few minutes at room tem perature To test for possible proteases mix the sample with SDS sample buffer without heating and leave at room temperature for 1 to 3 hr A loss of high molecular weight bands and a general smearing of the banding pattern indicate a protease problem Once heated the samples can sit at room temperature for the time it takes to load samples Carefully remove the Teflon comb without tearing the edges of the polyacrylamide wells After the comb is removed rinse wells with 1x SDS electrophoresis buffer The rinse removes unpolymerized monomer otherwise the monomer will continue to polymerize after the comb is removed creating uneven wells that will interfere with sample loading and subsequent separation Using a Pasteur pipet fill the wells with 1x SDS electrophoresis buffer If well walls are not upright they can be manipulated with a flat
419. ty can be converted to estimates of genetic similarity A convenient way to display this similarity data graphically is to generate a dendrogram with similar objects close to each other and less similar objects more distantly placed An example of such a dendro gram is presented in Figure 6 9 3 where sam ples from Listeria isolates are arranged based on banding pattern Current Protocols in Cell Biology Databases In many cases image analysis is not the last step in the process The image and analysis data need to be archived in a searchable format There may be a need to analyze the data from multiple experiments conducted at different sites or in laboratories around the world Bioin formatic links to diverse data sources might be desired to help develop a unified understanding of the biology behind particular phenomena When these situations arise database programs can be utilized to store link and search image analysis results As the number of images that are captured and analyzed grows it becomes increasingly more difficult to find particular information from the large number of files that are stored Relatively simple databases can be used if the major requirement is to find previously ana lyzed images and associated data Such data bases often display a miniaturized version of each image to aid in visual scanning for the file as well as simple searching for image specific information such as date of analysis file name
420. ubilization they should be stored at 80 C and should not be subjected to repeated freeze thawing Addition of SDS to the solubilization solution increases the solubilization of some proteins however SDS should only be used when the IEF gels contain urea and Triton X 100 as the solubilizing agents are required for effective separation of the strongly anionic SDS molecules from the proteins during isoelectric focusing Heating of samples in urea containing solutions must be avoided be cause urea readily decomposes to cyanate which reacts with amino groups and causes charge het erogeneity High concentrations of salts and buff ers in the sample should be avoided Ionic com pounds increase the conductivity of the sample and can result in localized overheating especially for Immobiline gels Electrophoresis and Immunoblotting 6 4 33 Supplement 4 Two Dimensional Gel Electrophoresis 6 4 34 Supplement 4 Samples analyzed on immobilized pH gradi ent gels should not contain precipitates The con centration of salts and buffer ions in the sample should be kept to a minimum lt 50 mM to avoid local overheating of the gel during electrophore sis If the sample forms aggregates or precipitates at the point of application apply the sample to a different location different pH on the gel or load the sample during the rehydration step Early decisions that must be made include the size of the gels required a
421. um proteins in a special square shaped free solution elec trophoresis cell His original work showed for the first time that protein components in serum could be separated giving the now fa miliar designations for a 8 and y globulin Arne Tiselius was awarded the Nobel Prize in Chemistry in 1948 http nobelprize org chemistry laureates 1948 index html for his research on electrophoresis and adsorp tion analysis especially for his discoveries concerning the complex nature of the serum proteins Electrophoresis is the movement of a charged particle including large molecules such as DNA and proteins in a liquid medium under the influence of an electric field The driving force QE on the charged molecule is a product of the charge Q and the electric field E across the separation gel Larger pro teins move more slowly as do proteins with a lower net charge Furthermore if a pro tein is in native compact form it will mi grate more quickly than the same protein fully denatured and extended where it experiences more frictional resistance with the surrounding medium Resistance in electrophoresis is de fined as f 6x rvn where fis the resistance of the medium to electrophoretic movement r the radius of the protein assumed to be a sphere v the electrophoretic velocity and 7 the vis cosity of the fluid The elements contributing to driving force and resistance together indi cate that protein charge size s
422. unoblotting 6 4 21 Supplement 4 BASIC PROTOCOL 4 Two Dimensional Gel Electrophoresis 6 4 22 Supplement 4 6 Retrieve isoelectric focusing gels containing protein samples to be analyzed from storage Incubate cryotubes containing frozen IEF gels in a 37 C water bath for 15 min for a 3 mm tube gel A 5 to 7 min incubation is sufficient for 1 5 mm or thinner IEF gels Do not agitate during thawing as vigorous agitation of a partially thawed gel can break the gel During this thawing equilibration step SDS in the equilibration solution in which the gels were frozen diffuses into the gel matrix and binds to proteins in the IEF gel The length of incubation in the equilibration buffer is critical because insufficient saturation of proteins with SDS will contribute to vertical streaks on staining On the other hand extended incubation in equilibration buffer will result in excessive loss of proteins owing to diffusion of protein out of the gel which is especially critical for thin IEF gels For this reason it is recommended that after extrusion from the IEF tube IEF gels be initially incubated for 5 min to allow adequate diffusion of glycerol into the gel to minimize gel breakage followed by freezing on dry ice see Basic Protocol 1 step 30 This is desirable even if the second dimension gel will be run directly after isoelectric focusing as it is the most feasible way of precisely controlling the equilibration time while t
423. upper surface of the gel Increase the constant current to 50 mA and run for an additional 3 to 4 hr or until the marker dye has migrated at least 6 to 7 cm Blotting the gel 13 Perform immunoblotting to a 0 45 um Immobilon P polyvinylidene fluoride PVDF membrane in a tank transfer system as described UNIT 6 2 with the exception of the following conditions a Electrophoretically transfer at a constant current of 100 mA at 4 C overnight b Use 0 25x transfer buffer without methanol c Use blocking buffer containing 5 w v nonfat dry milk Immunodetect protein 14 Perform immunoprobing with directly conjugated secondary antibody as described UNIT 6 2 except with the following variations which are specific for immunodetec tion of von Willebrand Factor protein a Dilute primary antibody rabbit anti vWF to a concentration of 1 4000 b Dilute secondary antibody donkey horseradish peroxidase linked anti rabbit Ig to a concentration of 1 2000 Visualization 15 Visualize von Willebrand factor protein on the PVDF membrane by meticulously following the recommendations enclosed in the ECL Western Blotting Analysis system Current Protocols in Cell Biology Electrophoresis and Immunoblotting 6 7 3 Supplement 15 ALTERNATE PROTOCOL Agarose Gel Electrophoresis of Proteins 6 7 4 Supplement 15 Visualization with Luminescent Substrates is discussed in UNIT 6 2 To determine the optimal concen
424. ver do not yield any information about the size number compo sition and relative abundance of MPCs A high resolution method that resolves these problems is Blue Native polyacrylamide gel electrophoresis BN PAGE Schagger and von Current Protocols in Cell Biology Jagow 1991 Schagger et al 1994 Originally it was developed by Hermann Schagger to separate mitochondrial membrane complexes in the mass range from 10 to 10 000 kDa Schagger and von Jagow 1991 Sch gger et al 1994 Later the protocol was modified for general applicability Camacho Carvajal et al 2004 Since the first description of BN PAGE descriptions of its use in pub lications has increased exponentially It has been applied successfully in nearly every area of multiprotein research e g purifi cation of complexes determination of their size Schagger et al 1994 Schagger 1995 Model et al 2001 Dudkina et al 2005 and stoichiometry Schamel et al 2005 Swamy et al 2007 protein complex assem bly Model et al 2001 structure determi nation by two dimensional crystallization and electron microscopy Poetsch et al 2000 identification of multiprotein complexes with mass spectroscopy Rexroth et al 2003 Camacho Carvajal et al 2004 Millar et al Electrophoresis and Immunoblotting al 6 10 17 Supplement 38 Two Dimensional Blue Native Polyacrylamide Gel Electrophoresis 6 10 18 Supplement 38 2005 or
425. versed in this procedure relative to equilibrium isoelectric focusing Omit steps 12 to 19 do not prefocus the gels 20 Remove the 8 M urea polymerization overlay solution and place 50 ul lysis buffer on top of each gel Wait at least 2 min then remove the lysis buffer 23 After loading the samples and overlaying with lysis buffer diluted with water 8 2 v v as in Basic Protocol 1 use 0 1 M H PO instead of NaOH to fill all gel tubes 24 Use 0 1 M H3PO as the upper electrode solution 25 Reverse the connection of electrodes i e connect the red lead to the upper chamber and the black lead to the lower chamber 26 Focus for a total of 3000 to 5000 Vhr The optimal number of volt hours depends on the nature of the sample and the ampholytes used The values recommended above may need to be adjusted empirically ISOELECTRIC FOCUSING USING IMMOBILIZED pH GRADIENT GEL STRIPS In immobilized pH gradient IPG gels the ampholytes are covalently linked to the acrylamide matrix which facilitates production of highly reproducible gradients as well as very narrow pH gradients for optimal resolution of minor charge differences A variety of precast gels and all the necessary equipment are commercially available from either Amersham Pharmacia Biotech or Bio Rad Equipment and chemicals are also available for the user to cast gels in the laboratory see Support Protocol 3 although precast gels are likely to suffice for the m
426. ving the comb while the agarose is still in a molten state and then reinserting often eliminates the formation of bubbles under the teeth of the comb 6 After the agarose has solidified place the apparatus at 4 C and allow the agarose to age 20 to 30 minutes The electrophoresis is to be carried out at 4 C The apparatus may either be placed in a cold room the electrophoresis buffer can be circulated through a refrigeration unit or the apparatus can be packed in wet ice 7 Overlay the solidified gel with 2 to 3 mm electrophoresis buffer 4 C 8 Remove the sample comb by lifting vertically in one smooth motion It is helpful to hold the gel down with the gloved fingers of the other hand to keep the gel from being pulled up when the comb is removed Prepare and load sample 9 Dilute the protein samples to twice the desired concentration using distilled water Immediately add the diluted sample to an equal volume of 2x sample buffer 10 Load a sample volume of 10 to 15 ul into the bottom of each sample well using a pipet with a fine tip or equivalent Prior to sample loading examine each sample well to ascertain that air bubbles are not trapped in the well Electrophorese gel also see UNIT 6 1 11 Run samples into the gel matrix at a constant current of 25 mA for 30 min or until the sample dye has completely entered the gel 12 Pause the electrophoresis and decrease the level of the electrophoresis buffer to 1 mm above the
427. wer supplies this is less of an issue However with age and use wires may become exposed through cracks in the insulation or poor connections Carefully inspect all cables and connections and replace frayed or exposed wires immediately 2 Always start with the power supply turned off Have the power supply controls turned all the way down to zero Then hook up the gel apparatus generally connect the red high voltage lead to the red outlet and the black high voltage lead to the black outlet Turn the power supply on with the controls set at zero and the high voltage leads connected Then turn up the voltage current or power to the desired level Reverse the process when the power supply is turned off i e to disconnect the gel turn the power supply down to zero wait for the meters to read zero turn off the power supply and then disconnect the gel apparatus one lead at a time CAUTION Jf the gel is first disconnected and then the power supply turned off a consider able amount of electrical charge is stored internally The charge will stay in the power supply over a long time This will discharge through the outlets even though the power supply is turned off and can deliver an electrical shock Ohm s Law and Electrophoresis Understanding how a gel apparatus is connected to the power supply requires a basic understanding of Ohm s law voltage current x resistance or V R A gel can be viewed as a resistor and the power supply as
428. wiches Do not add TEMED and ammonium persulfate until just before use 5 Fill a 50 ml syringe with the separating gel solution and slowly inject it into the caster until the gels are 6 cm high allowing 1 5 cm for the stacking gel 6 Overlay each gel with 100 ul H2O saturated isobutyl alcohol Allow the gels to ao polymerize for 1 hr Immunoblotting 6 1 25 Current Protocols in Cell Biology Supplement 37 SUPPORT PROTOCOL 3 One Dimensional SDS PAGE 6 1 26 Supplement 37 Pour the stacking gel 7 Remove the isobutyl alcohol and rinse with 1x Tris Cl SDS pH 8 8 Stacking gels can be cast one at a time with the gel mounted on the electrophoresis unit or all at once in the multiple caster 8 Practice placing a comb in the gel sandwiches before preparing the stacking gel solution Press the comb against the rectangular or taller plate so that all teeth of the comb are aligned with the opening in the gel sandwich then insert into the sandwich Remove combs after practicing 9 Prepare the stacking gel solution 2 ml per gel as directed in Table 6 1 1 Fill a 10 ml syringe with stacking gel solution and inject the solution into each gel sandwich 10 Insert combs taking care not to trap bubbles Allow the gels to polymerize 1 hr 11 Remove the front faceplate Carefully pull the gels out of the caster using a long razor blade to separate the sandwiches If the gels are left to polymerize for prolonged periods
429. with A B D or without C a UV white light conversion screen Bio Rad The image shown in C was acquired using an integration time of 0 6 sec in order to optimize sensitivity Current Protocols in Cell Biology Table 6 6 1 Troubleshooting Guide for Staining with Coomassie Blue Basic Protocol 1 Problem Cause Solution No bands are detected Background is completely clear Amount of protein s below Check protein concentration in original detection limit sample Stain the gel with silver Basic Protocol 2 Background is dark blue Insufficient destaining Continue to destain the gel until the background is clear steps 4 to 6 Protein bands are too faint Staining solution is too old i e Prepare new Coomassie blue staining methanol has evaporated solution Insufficient staining time Re stain the gel using a longer staining time step 2 Excessive destaining Re stain the gel monitoring band and background color during destaining steps 4 to 6 High background areas High background restricted to Interfering compounds in the Fix proteins with TCA before staining lanes with samples samples see Alternate Protocol 1 steps 2 to 5 Blue spots at the gel surface Powder or dirt deposited on the Use clean powder free gloves Remove surface during handling of the gel powder or dirt by gently touching the surface with a powder free glove Table 6 6 2 Troubleshooting Guide for Staining with Silver Basic Protocol 2 Pro
430. wo dimensional BN SDS PAGE of cellular lysates In this hypothetical experi ment total cellular lysates were prepared and dialyzed Support Protocol In A proteins were separated by BN PAGE Basic Protocol 1 and a subsequent second dimensional SDS PAGE Basic Protocol 2 Visualization of the protein was done with silver staining Monomeric proteins 3 4 and 5b were localized to a hyperbolic shaped diagonal indicating that they had the same size in the first and in the second dimension In contrast proteins that were present in the same multiprotein complex were found as individual spots aligned in vertical columns Complex 1 was a homotetrameric protein complex Complex 2 was composed of six proteins of small individual sizes Proteins 6 and 5 as well as 7 and 5 formed dimeric complexes In addition protein 5 was found as a monomer 5b Thus protein 5 existed in three different forms From the intensity of the spots one could deduce that complex 6 5 was more abundant than complex 7 5 In B the dialysed lysate was boiled with 1 SDS before separation by BN PAGE and SDS PAGE SDS destroys all multiprotein complexes thus the hyperbolic shaped diagonal can identified A similar wet experiment is described in Camacho Carvajal et al 2004 6 Heat the strips with the sample buffer in a microwave oven at medium power until the solution boils Let the gel strips shake for another 10 min at room temperature while cooling down
431. y of the protein near its isoelectric point and the separation distance from any near neighbors A variety of alternative gel sizes their limits and their advantages are summarized in Table 6 4 2 The lower protein limit for any of the systems is determined strictly by the available detection methods Proteins can be detected in two dimensional gels by the same wide range of techniques used for one dimensional gels Autoradiography UNIT 6 3 Silver staining and electroblotting to PVDF membranes unr 6 2 followed by colloidal gold or colloidal silver staining or immunodetection UNIT 6 2 are among the most sensitive techniques available Ifa larger amount of protein is available Coomassie blue staining of the gel or amido black staining of a PVDF membrane after electrotrans fer uni 6 2 would be the detection methods of choice The major technical limitation in two dimen sional gel electrophoresis is gel to gel variation Even when extreme care is exercised to produce highly reproducible first and second dimension gels some gel related variability among gels cast atthe same time is likely to persist Another source of variability includes differences in extraction or recovery of proteins during sample solubilization and handling Maximizing resolution and repro ducibility is especially important if computerized comparisons of two dimensional gels of complex protein mixtures such as cell or tissue extracts are being attempted
432. y during casting If the light solution is not flowing into the mixing chamber a bubble may be caught in the interconnecting valve Quickly close the outlet and cover the top of the reservoir chamber with a gloved thumb Push down with the thumb to increase the pressure in the chamber and force the air bubble out of the center valve 11 Fill the gel sandwich from the top Place the pipet tip against one side of the sandwich so the solution flows down one plate only The heavy solution will flow into the sandwich first followed by progressively lighter solution 12 Watch as the last of the light solution drains into the outlet tube and adjust the flow rate to ensure that the last few milliliters of solution do not flow quickly into the gel sandwich and disturb the gradient 13 Overlay the gradient gel with H2O saturated isobutyl alcohol Allow the gel to polymerize 1 hr In this gel system the gel will polymerize from the bottom i e heavy solution up Because polymerization is an exothermic reaction heat can be felt evolving from the bottom of the gel sandwich during polymerization A sharp optical discontinuity at the gel overlay interface indicates that polymerization has occurred In general I hr is adequate for polymerization 14 Remove the H2O saturated isobutyl alcohol and rinse with 1x Tris Cl SDS pH 8 8 Cast the stacking gel see Basic Protocol 1 steps 8 to 11 The gel can be covered with 1x Tris Cl SDS pH 8 8 sealed
433. y pour electrophoresis buffer into the upper reservoir taking care to avoid disturbing the agarose covered IEF gel 13 Connect electrodes and run the gels See UNIT 6 1 for electrophoresis conditions SECOND DIMENSION ELECTROPHORESIS OF IPG GELS In this protocol vertical gel electrophoresis is used as the second dimension for IPG gels in an analogous manner to the protocol described for the second dimension of IEF tube gels see Basic Protocol 3 One difference is the use of second dimension gel spacers or gel apparatus that will accommodate an 18 cm long Immobiline DryStrip Bio Rad offers a conversion kit to increase the gel width from 16 cm to 18 cm and Amersham Pharmacia Biotech offers the Iso Dalt gel system The use of beveled plates is not necessary as the 0 5 mm strips are narrower than the second dimension gel 1 0 or 1 5 mm thick Another change involves a two step equilibration of the strips prior to electrophoresis Current Protocols in Cell Biology Additional Materials also see Basic Protocol 3 DryStrip equilibration solutions 1 and 2 see recipes prepare fresh in step 4 Immobiline IPG DryStrip with focused protein see Basic Protocol 2 Platform shaker Cast the second dimension gel 1 Assemble the glass plate sandwich of an electrophoresis apparatus using gel plates wide enough to accommodate an 18 cm long DryStrip gel Beveled plates are not necessary If the spacers are not wide enough to accommodate an 18 cm
434. y to 3 mm IEF tube gels first dimension combined with 1 5 mm thick 16 x 16 cm size of separating gel second dimension gels see Basic Protocol 3 and may be easily adapted to a variety of different gel sizes see Table 6 4 1 A 3 mm IEF gel has a total protein capacity of 500 ug for complex protein mixtures such as whole cell extracts The maximum capacity of any single protein spot is 0 5 to 5 ug depending on the solubility of the protein near its isoelectric point and the separation distance from any near neighbors In this protocol gels are cast and prefocused before the sample is loaded The proteins are then separated according to isoelectric point and the gels are extruded from the tubes and stored Measuring pH profiles in IEF gels is a convenient and accurate method for determining pI see Support Protocol 1 To provide optimal reproducibility multiple gels should be cast and run simultaneously This is especially important for comparative studies involving complex mixtures of proteins The IEF gels may be cast either by pouring the gel solution into the gel tubes steps 3a to 7a or by using hydrostatic pressure steps 3b to 7b Pouring the gel solution into the gel tubes is convenient for 3 mm diameter IEF gels and requires only a minimal excess of reagents Because the gels are cast using a long needle and syringe for narrower gels where the needle does not fit inside the gel tube casting using hydrostatic pressure is more app
435. ycine gel buffer 10 10 10 10 10 10 10 H O 23 08 19 78 1648 12 98 9 78 646 3 18 10 w v ammonium 0 2 0 2 0 2 0 2 0 2 0 2 0 2 persulfate TEMED 0 02 0 002 0 02 002 0 02 0 02 0 02 Preparation of gel In a 75 ml side arm flask mix 30 acrylamide 0 8 bisacrylamide solution see Table 6 1 1 4x glycine gel buffer see Reagents and Solutions and H O If desired degas under vacuum 5 min to speed polymerization Add 10 ammonium persulfate and TEMED Swirl gently to mix Use immediately The recipes produce 40 ml gel solution which is adequate for one gel of dimensions 1 5 mm x 14 cm x 16 cm or two gels of dimensions 0 75 mm x 14 cm x 16 cm Pall reagents and solutions used in the protocol must be prepared with Milli Q purified water or equivalent Units of numbers in table body are milliliters The desired percentage of acrylamide in the gel solution depends on the molecular size of the protein being separated 4Must be freshly made e Added just before polymerization 10 Continue electrophoresis until the R marker reaches the bottom of the gel For minigels electrophoresis will take 1 to 2 hr Standard gels require 4 to 6 hr runs 11 Turn off power supply disassemble the unit and remove gel from sandwich 12 Stain the gel according to 4PPENDIX 3 NATIVE DISCONTINUOUS ELECTROPHORESIS AND GENERATION OF MOLECULAR WEIGHT STANDARD CURVES FERGUSON PLOTS One straightforward approach to discontinuous nat
436. ying screens are used to enhance the film image generated by radioactive mole cules Laskey and Mills 1977 Laskey 1980 They are used strictly in conjunction with strong B emitting isotopes such as P or y emitting isotopes such as 1I Emissions from these forms of radiation will frequently pass completely through a film but they can be absorbed by an intensifying screen which fluoresces and exposes the film with multiple photons of light While an intensifying screen will substantially enhance the film image as compared with direct exposure Table 6 3 1 some loss of image resolution will occur due to light scatter Intensifying screens are distributed by most laboratory supply companies e g Fisher Sigma and Kodak As shown in Figure 6 3 1 the film should be placed between the sample and the intensifying screen Preflashed film see Support Protocol 3 should be used if the sample is weakly radioactive or if quantitation of the radioactivity is desired The preflashed side of the film should be placed adjacent to the intensifying screen For very weakly radioactive samples a second screen can be placed on the other side of the radioactive sample i e screen then sample then film then screen but this causes further loss in resolution due to light scatter Also the sample and sample support must be sufficiently transparent to allow light from the second screen to reach the film The film should be exposed at 70 C to stabilize the
437. ystem or a modified Tris buffer in the absence of urea Continuous SDS PAGE is a simplified method in which the same buffer is used for both the gel and the electrode solutions and the stacking gel is omitted Other protocols cover the preparation and use of ultrathin gels and gradient gels and the simultaneous preparation of multiple gels Curr Protoc Cell Biol 37 6 1 1 6 1 38 2007 by John Wiley amp Sons Inc Keywords protein e electrophoresis e separation e polyacrylamide e SDS PAGE INTRODUCTION Electrophoresis is used to separate complex mixtures of proteins e g from cells sub cellular fractions column fractions or immunoprecipitates to investigate subunit com positions and to verify homogeneity of protein samples It can also serve to purify proteins for use in further applications In polyacrylamide gel electrophoresis PAGE proteins migrate in response to an electrical field through pores in the gel matrix pore size decreases with higher acrylamide concentrations The combination of gel pore size and protein charge size and shape determines the migration rate of the protein The standard Laemmli method see Basic Protocol 1 is used for discontinuous gel electrophoresis under denaturing conditions that is in the presence of sodium dodecyl sulfate SDS The standard method for full size gels e g 14 x 14 cm can be adapted for the minigel format e g 7 3 x 8 3 cm see Basic Protocol 2 Minigels provide rapi
438. ze decomposition of urea If the reswelling tray is used 250 or 400 ul rehydration solution is required per 11 or 18 cm DryStrip respectively bA total urea concentration of 9 M is typically used Thiourea is more effective than urea for minimizing protein precipitation during isoelectric focusing but its solubility is lower The combination of 7 M urea 2 M thiourea usually results in superior sample solubilization and isoelectric focusing as compared with 9 M urea alone The optimal detergent and detergent concentration should be empirically determined Other common alternatives are Triton X 100 and octyl glucoside The detergent used must be nonionic or zwitterionic to avoid high current and consequent overheating during isoelectrofocusing Alternatively soluble ampholytes are available as preformulated IPC buffer concentrates for each type of IPG strip Thoroughly clean all equipment with a mild laboratory detergent solution rinse well with Milli Q water and allow to dry before using Solutions containing 10 M urea may be heated briefly to 30 to 40 C to aid in solubilization Materials Urea ultrapure CHAPS or Triton X 100 Pharmalyte 3 10 4 6 5 and or 8 10 5 soluble ampholytes see Table 6 4 1 Amersham Pharmacia Biotech Ampholine pH 6 8 Amersham Pharmacia Biotech DTT dithiothreitol Bromphenol blue Precast Immobiline DryStrips Amersham Pharmacia Biotech DryStrip cover fluid Amersham Pharmacia Biotech Immob

"Electrophoresis and Immunoblotting". In: Current Protocols in Cell

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&quot;Electrophoresis and Immunoblotting&quot;. In: Current Protocols in Cell

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"Electrophoresis and Immunoblotting". In: Current Protocols in Cell