PROTEAN® II xi Cell and PROTEAN II xi 2
Contents
1. 16 cm Set one left one right 20 cm Set one left one right Optional and Replacement Parts Central Cooling Core with gaskets Lower Buffer Chamber Cell Lid with safety cables Upper Buffer Dam 1 Slab Casting Stand with gaskets Replacement Gaskets for casting stand 2 Gradient Pouring Needle for bottom filling 2 Replacement Gaskets central cooling core 2 Alignment Card 34 165 1841 165 1846 165 1842 165 1847 165 1843 165 1848 165 1844 165 1849 165 1845 165 1850 165 1901 165 1902 165 1806 165 1807 165 1808 165 1909 165 1911 165 1912 165 2007 165 1913 165 2029 Catalog Product Description Number Combs each 25 well x 0 75 mm 60 ul volume 165 1861 25 well x 1 0 mm 80 ul volume 165 1862 25 well x 1 5 mm 120 ul volume 165 1863 20 well x 0 5 mm 54 ul volume 165 1865 20 well x 0 75 mm 82 ul volume 165 1866 20 well x 1 0 mm 110 ul volume 165 1867 20 well x 1 5 mm 164 ul volume 165 1868 20 well x 3 0 mm 328 ul volume 165 1869 15 well x 0 5 mm 74 ul volume 165 1870 15 well x 0 75 mm 110 ul volume 165 1871 15 well x 1 0 mm 147 ul volume 165 1872 15 well x 1 5 mm 221 ul volume 165 1873 15 well x 3 0 mm 442 ul volume 165 1874 10 well x 0 5 mm 114 ul volume 165 1875 10 well x 0 75 mm 172 ul volume 165 1876 10 well x 1 0 mm 229 ul volume 165 1877 10 well x 1 5 mm 343 ul volume 165 1878 10 well x 3 0 mm 687 pl volume 165 1879 5 well x 1 0 mm 522 ul volume 165 1882 5 well x 1 5 mm 7832. NNN SS SO SS NNN NNN NNN SS SS SO SS SS SS SS SO SO SS SO SO SS SS A ANNA DANAAAANI PROTEAN II xi Cell and PROTEAN II xi 2 D Cell Instruction Manual For Technical Service Call Your Local Bio Rad Office or in the U S Call 1 800 4BIORAD 1 800 424 6723 NOSON SS SO SS SS SS SS SS SS SS SS SO SS SS SS SS SN SS SS SS SS SO SS SS SS SS SS SS SSSI SS SO SSSI Section 1 1 1 1 2 13 Section 2 2 1 2 2 2 3 2 4 2 5 2 6 2 2 8 Section 3 3 1 3 2 Section 4 4 1 4 2 4 3 4 4 Section 5 5 1 5 2 Section 6 6 1 6 2 6 3 Section 7 Section 8 8 1 8 2 Section 9 Section 10 10 1 10 2 Section 11 Section 12 Section 13 13 1 13 2 13 3 13 4 13 5 Table of Contents Page General Infurmatton ALI FN 1 Non 06 864 1c EEN 1 SPECITICATLONS E 1 SSH NE HE YR FFR NF NFYFC FYNENT FFF FYFYR 2 Description of Major Parts 3 Central Cooling Core maamassa gn DY GR E TY EEF ION 3 Sandwich CaM Psi een ie RYG Tu Nyd YN FF el YF FI Y EN 3 EE EE 3 Upper Buffer Chamber i eT 1 aal CY GG 4 Lower Butler Chamber ei si FF Ed a an e EN i ee 4 Ee DEE A Tube Gel Ada Ptn eiseirge ir aren FFO DY sg GRY CY Ro GYD FFA NF aN 4 Alsnment Cardis EE 4 Assembling the Glass Plate Sandwiches isie 4 Assembling Single Sandwiches Ynn HYLL a n a EL LL nL nana aan aaeaen 4 Assembling Multiple or Double up Gel Sandwiches eee eee eerste 8 Casting the E DEE 9 Casting Discontinuous Laemmli Gel 9 Casting Continuous Gel Si
3. er sealing when solution is poured Tighten the other clamp screw in the same manner It is important to visually inspect the sandwich while it is in the alignment slot to insure that there are no gaps between the glass plates and the surface of the alignment slot 9 Remove the alignment card Pull the gel sandwich from the alignment slot Check that the plates and spacers are flush at the bottom If not realign the gel sandwich as in steps 6 8 Note The easiest way to check for proper alignment is to run a fingernail across the con tact area between the glass plates and spacer If your fingernail catches or drops as you move from plate to spacer to plate you must realign the sandwich before proceeding to step 10 10 The cams on the casting stand should be handle up and pulled out Place the aligned sand wich into one of the casting slots with the longer plate facing you and the arrows on the clamp facing away from you When the sandwich is placed correctly push the cams in and turn them 180 so that the handles of the cams point downward The sand wiches are now ready for gel casting 3 2 Assembling Multiple or Double up Gel Sandwiches Up to four gels can be run at one time by doubling up gel sandwiches i e 2 gels side Double gels are assembled aligned and cammed in a manner very similar to that described for single gels Note In order to run four gels instead of two it is necessary to order two notched i
4. Clin Med 68 842 1966 Margolis J and Kendrick K G Anal Biochem 25 347 1968 Ornstein L and Davis B J Ann N Y Acad Sci 121 321 1964 Reisfeld R A Lewis U J and William D E Nature 195 281 1962 Blattler D P Garner F Van Slyke K and Bradley A J Chromatog 64 147 1972 Jeppesen P G N Anal Biochem 58 195 1974 15 3 SDS Gel Systems References 1 2 3 4 9 6 7 8 9 Shapiro A L Vinuela E and Maizel J V Biochem Biophys Res Commun 28 815 1967 Laemmli U K Nature 227 680 1970 Fairbanks G Steck T L and Wallace D F H Biochemistry 10 2606 1971 Weber K and Osborn M J Biol Chem 224 4406 1969 Yamada K M and Weston J A Proc Nat Acad Sci U S A 71 3492 1974 Neville D M J Biol Chem 246 6328 1971 O Farrell P Z Gold L M and Huang VV M J Biol Chem 248 5499 1973 Studier F W J Mol Biol 79 237 1973 Ferro Luzzi Ames G J Biol Chem 249 634 1974 15 4 Urea Gel Systems References ES eg Kaltschmidt E and Wittmann H G Anal Biochem 36 401 1970 Swank R W and Muckers K D Anal Biochem 39 462 1971 Jakes K Zinder N D and Boon T J Biol Chem 249 438 1974 Mets L J and Bogorad L Anal Biochem 57 200 1974 Sherton C C and Wool I G J Biol Chem 249 2258 1974 15 5 Two Dimensional IEF SDS PAGE Gel Syste
5. Distilled water 3 8 ml 0 5 M Tris HCl pH 6 8 1 0 ml Glycerol 80 ml 1096 w v SDS 1 6 ml 2 B mercaptoethanol 0 4 ml 0 05 w v bromophenol blue 0 4 ml 8 0 ml Dilute the sample at least 1 4 with sample buffer and heat at 95 C for 4 minutes 5X Electrode Running Buffer pH 8 3 enough for 10 runs Tris base 45 g 15 g l Glycine 216 g 72 g l SDS 15 g 5 g l to 3 liters with dH O Store at 4 C Warm to 37 C before use if precipitation occurs Dilute 300 ml 5X stock with 1 200 ml dH O for one electrophoresis run Do not adjust the pH vith acid or base Laemmli U K Nature 227 680 1970 39 14 2 Separating Gel Preparation 0 375 M Tris pH 8 8 1290 7 5 Distilled water 33 5 ml 48 5 ml 1 5 M Tris HCl pH 8 8 25 0 ml 25 0 ml 10 w v SDS stock store at room temperature 1 0 ml 1 0 ml Acrylamide Bis 30 stock Degas for 215 minutes at room temperature 40 0 ml 25 0 ml 10 ammonium persulfate fresh daily 500 ul 500 ul 0 05 TEMED 50 ul 50 ul 0 05 TOTAL MONOMER 100 ml 100 ml To make the 10 ammonium persulfate solution dissolve 100 mg APS in 1 ml dH O One can prepare any desired volume of monomer solution by using multiples of the 100 ml recipes a For SDS treated proteins in the approximate molecular weight range between 10 and 100 K Daltons Use Bio Rad s Low MW Standards catalog number 161 0304 for 12 separating gel b For SDS treated proteins in the approximate mol
6. Make sure the core caps are in place on the cooling core ports 2 Seat the white U shaped gasket onto the core with the flat non stepped side down Note To help insure a good upper buffer seal with your gaskets for the PROTEAN II xi cell lubricate the entire front of the gaskets the shaded portion with water or upper buffer prior to attaching the gel sandwich to the central cooling core This will allow the glass plate sandwich to slide onto the gasket properly 3 After the gel is cast and the comb is removed if applicable release the gel sandwich from the casting stand by turning the cams to the up position and pulling them out ward Pull the gel sandwich straight out of the stand 15 4 With the short glass plate facing the cooling core and the locating decal on the clamps facing the core position the gel sandwich so that the grooves in the upper portion of the clamps are fitted onto the locating pins on the central cooling core The gel sand wich should be positioned at an angle of lt 20 with the cooling core Keeping this angle to a minimum and lubricating the gasket will prevent distortion of the gasket while the sandwich slides into place Note The locating pins on the central cooling core must be tightly secured in place to insure optimal pressure during operation If these pins are vibrated loose during transport or repeated use the decrease in pressure on the clamp can be enough to allow the c
7. 2 cm below the teeth of the comb This will be the level to which the separating gel is poured Remove the comb Add APS and TEMED to the deaerated monomer solution and pour the solution to the mark using a glass pipet and bulb The easiest way to pour is to flow the solution down the middle of the outside plate of the gel sandwich Another way to pour is to flow the solution down the side of one of the spacers An alternative method is to use a syringe and Tygon tubing to load the solution from near the bottom of the sandwich In all cases pour the solution smoothly to prevent it from mixing with air r a Immediately overlay the monomer solution with water water saturated isobutanol or t amyl alcohol The advantage of using isobutanol or t amyl alcohol is that the overlay solution can be applied rapidly with a Pasteur pipet and bulb because very little mix ing will occur If water is used to overlay it must be done using a needle and syringe using a steady even rate of delivery to prevent mixing Allow the gel to polymerize for 45 minutes to 1 hour Rinse off the overlay solution com pletely with distilled water This is especially important with alcohol overlays Do not allow alcohols to remain on the gels more than 1 hour or dehydration of the top of the gel will occur Note It is sometimes convenient to cast the separating portion of a discontinuous gel the afternoon before casting the stacking gel and running the gel If the stac
8. 6 for ordering information Current Conditions mA per gel Gel Thickness Stacking Gel Separating Gel 0 5 8 12 0 75 13 18 1 0 16 24 1 5 25 35 3 0 50 70 Run time is between 4 and 5 hours depending on length of gel Under constant current conditions voltage will gradually increase during the run As a safety precaution always set voltage current and power limits when possible Recommended power conditions for optimal resolution with minimal distortion 41 14 5 Comparison of Coomassie Blue and Silver Staining Coomassie Blue Silver Stain Procedure Sensitivity Stain 1 2 hour vvith 0 190 Coomassie Blue R 250 in fixative 4090 MeOH 10 HOAc Destain with 40 MeOH 10 HOAc to remove background usu ally 1 to 3 hrs 14 6 2 D Stock Solutions First Dimension IEF Tube Gels Detergent Solution 0 3 g CHAPS 100 ul Nonidet P 40 900 ul ddH20 Dissolve CHAPS in water then add Nonidet P 40 NP 40 First Dimension Capillary Tube Gel Monomer Solution 11 g urea 9 2 M final concentration 3 ml acrylamide bis stock 4 5 total monomer Stir to dissolve the urea This step is done slowly without heating or with very gentle heating only 0 2 ml Bio Lyte 5 7 ampholyte 0 8 ml Bio Lyte 3 10 ampholyte 1 ml detergent solution CHAPS NP 40 from above Add deionized water to 20 ml Mix and degas Add 20 ul TEMED after degassing to avoid evaporation Add 40 ul 10 w v APS swirl 8 10 times and cast the gels T
9. agarose overlay is 1 agarose in 1x stacking gel buffer diluted 1 4 see Section 14 1 solution C Note If you would like to apply molecular weight standards to the second dimension we recommend using a 2 D comb for casting the stacking gel As an alternative if a stack ing gel is not desired you can make a tube gel with a mixture of agarose and Bio Rad standards Then simply cut the agarose into pieces and load a piece directly on top of the second dimension slab gel in tandem with the IEF tube gel 4 Electrophorese the SDS slab gel as in Section 7 5 Remove the gels as in Section 9 6 Stain the gels as in Section 14 5 Note Tube gels may be frozen for future use or applied directly to a second dimension slab gel To freeze a tube gel place the gel lengthwise in a stoppered tube in an EtOH dry ice bath Once frozen gels can be stored at 20 C 29 Section 11 Maintenance of Equipment PROTEAN II xi cell chamber core Rinse thoroughly with distilled water clamps after every use Glass plates spacers combs Wash with a laboratory detergent cata log number 161 0722 then rinse thor oughly with distilled water Glass plates if more stringent cleaning Soak in a strong acid solution chromic is required acid sulfuric acid cleaning solution for 230 minutes and then rinse thoroughly with distilled water A less toxic alterna tive is 5 KOH in 100 methanol Glass tubes After use rinse with laboratory detergent s
10. first dimension gels at room temperature with a constant voltage of 200 volts for 2 hours followed by 500 volts for 2 hours and then 800 volts overnight 16 hours As a safety precaution always set voltage current and power limits when possible Note Phycocyanin is a colored protein found in Bio Rad s IEF standards catalog number 161 0310 Although these standards cannot be used for pI calibration in 2 D procedures because denaturation in urea produces too many peptide spots the phyco cyanin is excellent for monitoring the first dimension IEF It retains its blue color and will focus in a tight blue band when focusing is finished Loading one tube with the focus ing standards is an easy way to monitor the progress of the focusing run Cast the second dimension SDS slab gel during the running of the first dimension see Section 4 Note This protocol does not use a stacking gel However if a stacking gel is required for a particular application it should be cast on a level surface It is important that the same amount of monomer be used for each stacking gel to insure stacking gels of iden tical depth If a comb is not used as in most 2 D applications then the stacking gel should be overlaid with 1 0 ml of water saturated sec butanol After polymerization is complete drain off the overlay or remove the combs and rinse the gel surface briefly with dis tilled deionized water Extruding Tube Gels 1 After electrophoresis
11. for 20 cm so that you can insert the pipet tip fur ther into the well before sample delivery This will prevent inter well mixing of samples Or use the Bio Rad Prot Elec tips catalog number 223 9915 or 223 9917 which are designed for loading samples into wells 6 2 Loading a Single Sample Per Gel In this procedure a gel is cast without using a comb forming a flat gel surface This gel is cast with an overlay solution This type of sample application can be used for preparative purposes but it is most often used in blotting applications After electrophoresis the sam ple is transferred electrophoretically to a membrane which then can be cut into several identical strips for analysis Follow the instructions for casting the separating portion of a discontinuous gel Section 4 1 except pour the gel nearly to the top of the sandwich Allow just enough room for sample loading A stacking gel may also be added to this type of gel 1 Prepare electrode buffer and add to lower reservoir as in Section 6 1 Place the central cooling core in the lower chamber and add electrode buffer to the upper reservoir cham ber 2 The sample may be loaded with an Eppendorf type pipettor with a needle and syringe or with a Hamilton syringe Start at one end of the gel and deliver the sample gently and evenly over the entire length of the gel Layer the sample as closely as possible 1 2 mm to the surface of the gel 6 3 Gels as Samples All two
12. liters for 20 cm plates while providing excellent heat exchange through the central cooling core 2 6 Lid Combined with the lower buffer chamber the lid acts to fully enclose the PROTEAN II xi cell during electrophoresis thus providing electrical insulation The lid cannot be removed without disconnecting the electrical circuit It can be placed on the lower cham ber in only one alignment so that the anode and cathode connections cannot be accidentally reversed The lid also holds the coiled electrical leads when not in use 2 7 Tube Gel Adaptor The tube gel adaptor clamps onto the central cooling core in one easy motion and pro vides a leak proof seal for the upper buffer chamber at voltages up to 1 000 V especially useful for 2 D applications The molded construction produces a lightweight yet durable adaptor unit which has a gel tube locator at the bottom to hold the tubes in a vertical ori entation Each adaptor can hold up to 8 tubes from 1 0 mm ID to 6 mm ID 16 tube gels can be run at once using two tube adaptors Note The upper buffer dam may not be used opposite a tube gel adaptor 2 8 Alignment Card The alignment card greatly simplifies sandwich assembly by keeping the spacers upright during sandwich alignment A sandwich is assembled by placing two spacers on top of the large outer plate The alignment card is placed between the two spacers and the shorter inner plate is then placed on top Following attachment of t
13. plastic sheets 5 Pipet 150 ml of transfer solution along the length of the extruded gel Ki 28 Embedding of First Dimension onto Second Dimension Note VVhen handling gels it is advisable to vvear gloves First dimension gels tend to be sticky to the touch and will tear easily 1 Use of a beveled short glass plate catalog number 165 1827 for 16 cm cell and 165 1828 for 20 cm cell in the second dimension slab sandwich will greatly improve the ease of embedding the tube gel on top of the slab gel Rinse the top of the completed second dimension SDS slab gel thoroughly with distilled water and drain off excess water Attach clamp assemblies to cooling core 2 Using a spatula direct the gel from the Parafilm to the bevel at the top of the inner glass plate starting at one side and proceeding across the gel Place a few drops of SDS electrode buffer along the top of the tube gel 3 Use a spatula to seat the tube gel on the slab gel Check that the tube gel is in contact with the slab gel over its entire length Be sure to remove all air bubbles that are between the tube gel and the slab gel By placing the tube gel directly on top of the second dimension slab gel between the glass plates no agarose overlay is necessary If the tube gel diameter is greater than the second dimension slab gel thickness an agarose over lay may be necessary to insure good contact and to prevent the tube gel from slipping off the slab gel The
14. remove the tube gels from the tube gel adaptor Rinse both the top and bottom of each gel thoroughly with distilled water Failure to rinse before extrusion will result in residual base NaOH or acid phosphoric on the gel that will interfere with measurement of the pH gradient Place the tubes in order into the tube rack and fill each tube to the top with distilled water Attach a long at least 2 inch fine at least 26 gauge beveled needle such as catalog number 165 1944 to a3 ml plastic syringe Rim the upper and lower few mms of the gel by inserting the needle between the gel and the glass tube point against the glass wall to prevent tearing of the gel while forcing distilled water through the syringe and needle Turn the gel tubes so that the entire circumference is rimmed see photo Often tube gels may be extruded without rimming using water pressure as below 27 3 Attach a piece of silicone tubing to a ml syringe and to the outside wall of the glass tubes on the top end of the tube Using the syringe filled with distilled water apply a firm even pressure to start the gel extruding from the tubes As the gel moves further out of the tube apply less pressure so that rate of extrusion remains constant Care must also be taken not to extrude too quickly Only slight pressure is required to remove the last 1 2 cm of gel from the tube 4 It is convenient to extrude the gels onto longitudinally folded pieces of Parafilm or
15. ul volume 165 1883 5 well x 3 0 mm 1 57 ml volume 165 1884 3 well x 1 5 mm 1 37 ml volume 165 1888 Blank x 0 75 mm 2 44 ml volume 165 1891 Blank x 1 0 mm 3 26 ml volume 165 1892 Blank x 1 5 mm 4 88 ml volume 165 1893 Blank x 3 0 mm 9 76 ml volume 165 1894 2 D x 1 0 mm ref well 28 ul ref well volume 165 1897 2 D x 1 5 mm ref well 42 ul ref well volume 165 1898 2 D x 3 0 mm ref well 84 ul ref well volume 165 1899 Teflon Comb Conversion Screws 10 165 1859 To convert any PROTEAN II xi comb with a standard 25 mm well depth to a 10 cm well depth May be used for agarose or acrylamide gels 35 Glass Tubes Product Description ID OD Length Pkg Catalog mm mm mm Qty Number 1 0 5 0 180 24 165 3136 1 5 6 0 150 24 165 3137 1 5 6 0 180 24 165 3138 2 0 6 5 180 24 165 3139 2 4 4 0 160 24 165 3155 3 0 5 0 125 24 165 3150 5 0 7 0 125 24 165 3122 13 3 PROTEAN ll xi 2 D Cells Catalog Product Description Number PROTEAN II xi 2 D Cell 1 0 mm 16 cm 165 1931 PROTEAN II xi 2 D Cell 1 5 mm 16 cm 165 1932 PROTEAN II xi 2 D Cell 1 0 mm 20 cm 165 1933 PROTEAN II xi 2 D Cell 1 5 mm 20 cm 165 1934 PROTEAN II xi 2 D cells include central cooling core lower buffer chamber lid with power cables 2 sets of glass plates with bevel 4 sandwich clamps 24 glass tubes tube diameter spacer thickness 2 tube gel adaptors 16 stoppers 16 grommets 2 2 D combs 4 spacers 1 upper buffer dam slab casting sta
16. 4 C for buffer and monomer stocks If protein bands are diffuse as well as to the tracking dye increase current by 25 50 and or increase T of resolving gel Dilute sample selectively remove predominant pro tein in the sample or reduce current by about 25 to minimize streaking Centrifuge sample or decrease T of resolving gel If urea nonionic detergent is not sufficient use SDS as in Ref 15 5 2 Centri fugation of sample may be necessary up to 100 000 x g for 30 minutes to remove undissolved particulates Treat sample with DNase or RNase as in Ref 15 5 1 Increase current by 20 Deionize urea Minimize the time between sample application and power start up Increase T of stacking gel to 4 5 or 5 T or increase current by 25 during stacking Troubleshooting Guide continued Problem Cause Solution 7 Skewed or distorted bands a Poor polymerization around a Degas stacking gel solution sample wells thoroughly prior to casting increase ammonium per sulfate and TEMED con centrations by 25 or add riboflavin phosphate to 5 ug ml in addition to the usual catalyst levels in the stacking gel b Salts in sample b Remove salts by dialysis desalting column etc c Uneven gel interface c Increase reaction rate over lay carefully 8 Run taking unusually a Buffers too concentrated a Check buffer protocol long time dilute if necessary b Low current b Increase curre
17. 4 6 Ampholyte 40 25 ml 163 1143 Bio Lyte 5 7 Ampholyte 40 25 ml 163 1153 Bio Lyte 5 8 Ampholyte 40 25 ml 163 1193 Bio Lyte 6 8 Ampholyte 40 25 ml 163 1163 AG 501 X8 Mixed Bed Ion Exchange Resin 100 g 143 7424 13 6 Power Supplies Catalog Product Description Number PowerPac 3000 100 120 VAC 165 5056 PowerPac 3000 220 240 VAC 165 5057 Model 1000 500 Power Supply 100 120 VAC 165 4710 Model 1000 500 Power Supply 220 240 VAC 165 4711 38 Section LA Appendix 14 1 Reagents and Gel Preparation for SDS PAGE Slab Gels Laemmli buffer system Stock Solutions A Acrylamide Bis 30 T 2 67 C 146 g acrylamide 29 2 g 100 ml 4 g N N Bis methylene acrylamide 0 8 g 100 ml Make to 500 ml with distilled water Filter and store at 4 C in the dark 30 days maximum Or substitute Bio Rad s Preweighed Acrylamide Bis 37 5 1 mixture Catalog No 161 0112 30 g Catalog No 161 0106 200 g 150 g Acrylamide Bis 30 g 100 ml to 500 ml with dH O 1 5 M Tris HCl pH 8 8 54 45 g Tris base 18 15 g 100 ml 150 ml distilled water Adjust to pH 8 8 with 1 N HCI Make to 300 ml with distilled water and store at 4c 0 5 M Tris HCl pH 6 8 6 g Tris base 60 ml distilled water Adjust to pH 6 8 with 1 N HCI Make to 100 ml with distilled water and store at 4 C 10 SDS Dissolve 10 g SDS in water with gentle stirring and bring to 100 ml with dH O Sample Buffer SDS reducing buffer store at room temperature
18. 4 7 Section 15 15 1 15 2 15 3 15 4 15 5 OO KEE 39 Reagents and Gel Preparation for SDS PAGE Slab GelS 39 Separating Gel Preparation 00 0 0 eee eeeeceeeeseceeeeeeeseecaecaeecaecacsaecaeeeenseeseees 40 Stacking Gel Preparation ue i ieu reed ss FF Dnd dT yW kea a ro s 41 Running Conditions ossia YF I RT tse DYU REDD YF UD 41 Comparison of Coomassie Blue and Silver Staining eo022o2220eeoneo seca 42 DEP StOCK SOMOS cie iii EES 42 Running Conditions scssi ns chy oth as E ae EEE SEEE E S 43 Appendix jin Ee AGS eae en eae 44 General References eege esd esis soe esti tetes ritmes a estrenes ES 44 Native Gel Systems References uuuosuussn nnen ean e ae e aa eee enaa 44 SDS Gel Systems Relerences m ssssms m keen mivat tes e 44 Urea Gel Systems References ennnen aa a a aa na aa a naene ean 44 Two Dimensional IEF SDS PAGE Gel Systems References 44 Note To insure best performance from the PROTEAN II xi cell become fully acquainted vvith these operating instructions before using the cell to separate samples Bio Rad recommends that you first read these instructions carefully Then assemble and disassemble the cell completely without casting a gel After these preliminary steps you should be ready to cast and run a gel Bio Rad also recommends that all PROTEAN II xi cell components and accessories be cleaned with a suitable laboratory cleaner such a
19. 61 0610 Dithiothreitol 5g 161 0611 2 mercaptoethanol 25 ml 161 0710 TEMED 5ml 161 0800 TEMED 50 ml 161 0801 Agarose Standard Low m 100 g 162 0100 CHAPS lg 161 0460 CHAPSO lg 161 0465 Agarose Standard Low m 500 g 162 0102 SDS PAGE Standards 14 400 97 400 MW 161 0304 SDS PAGE Standards 45 000 200 000 MW 161 0303 Prestained SDS PAGE Standards low range 161 0305 Prestained SDS PAGE Standards high range 161 0309 2 D SDS PAGE Standards 161 0302 Silver Stain SDS PAGE Standards low range 161 0314 Silver Stain SDS PAGE Standards high range 161 0315 Silver Stain Plus Kit includes fixative enhancer concentrate 161 0449 silver complex solution reduction moderator solution image development reagent development accelerator reagent complete instructions and Material Safety Data Sheets 37 13 5 Electrophoresis Chemicals continued Quantity per Catalog Product Description Package Number Coomassie Blue R 250 10 g 161 0400 Bromophenol Blue 10 g 161 0404 Triton X 100 500 ml 161 0407 Urea 250 g 161 0730 Urea 1 kg 161 0731 Bio Lyte Ampholytes Bio Lyte 3 10 Ampholyte 40 10 ml 163 1112 Bio Lyte 3 5 Ampholyte 20 10 ml 163 1132 Bio Lyte 4 6 Ampholyte 40 10 ml 163 1142 Bio Lyte 5 7 Ampholyte 40 10 ml 163 1152 Bio Lyte 6 8 Ampholyte 40 10 ml 163 1162 Bio Lyte 7 9 Ampholyte 40 10 ml 163 1172 Bio Lyte 8 10 Ampholyte 20 10 ml 163 1182 Large Volume Ampholytes Bio Lyte 3 10 Ampholyte 40 25 ml 163 1113 Bio Lyte
20. AN II xi 2 D cell including tube adaptor 1 Lower buffer chamber 2 Lid with elec trical leads in place 3 Cooling core with glass plate sandwich attached and core caps in place 4 Casting stand with glass plate sandwich in alignment slot 5 Tube gel adaptor 6 Sandwich clamps 7 Buffer dam 8 Alignment card 2 1 Central Cooling Core The central cooling core provides the cooling capability which prevents thermal band distortion during electrophoretic separations The cooling core can be connected to any circulating cooling source The serpentine flow pattern assures even heat distribution over the entire gel area An ethylene glycol water 20 80 solution is recommended as coolant Other coolants may damage the plastic during extended exposure If a circulating bath is not available the core can be connected to a tap water line or simply filled with 1 8 liters of coolant and plugged to act as a heat sink during electrophoresis The central cooling core has a raised upper electrode which is housed in a protective cas ing and the lower electrode is recessed to prevent accidental damage 2 2 Sandwich Clamps The unique PROTEAN H xi sandwich clamps consist of a single screw mechanism which makes assembly alignment and disassembly of the gel sandwich an effortless task The clamps exert an even pressure over the entire length of the glass plates providing a leak proof seal and preventing plate damage due to uneven pressure Each pair of c
21. arely down on a piece of Parafilm to remove excess acrylamide Wipe off the excess acrylamide Inspect the gels before loading bubbles within the gel prevent focusing and these gels should be discarded Note Alternative methods for filling capillary tubes can be used such as wrapping the bottom end of the capillary tube with two layers of Parafilm laboratory film and filling using a syringe and fine gauge cannula gel tube loading needle 165 1943 The cannula should be long enough to reach the bottom of the tube Slowly inject the acry lamide solution into the bottom of the tube withdrawing the cannula as the acrylamide enters the tube Fill to the mark on the tube Sample Preparation and Loading Sample preparation prior to isoelectric focusing is one of the most important steps for obtaining reproducible two dimensional electrophoresis gels There is no method which is optimal for every sample and it may be necessary to experiment with different protein sol ubilization methods to determine which is best 1 2 Prepare the first dimension running solutions as described in Section 14 Prepare the IEF sample concentrate solution A and or iso urea solution E as described in Section 14 These solutions should be prepared fresh or frozen in aliquots Note Sample loads above 400 ug total protein may cause loss of resolution in the sec ond dimension slab gel Replace the notched white gaskets with the tube adaptor gaskets s
22. casting loading and running tube gels 23 10 1 Sequence of Steps for 2 D Protocol Step Time Interval Day 1 k Pour4ubegels it Neil polymerize 2 hours 2 Prepare electrolytes prepare and load samples 4 hour 1 hour 3 Electrophorese at 200 V constant voltage 2 hours 4 Electrophorese at 500 V constant voltage 2 hours 5 Electrophorese at 800 V constant voltage 16 hours overnight 6 Cast slab gels for second dimension gels while first dimension gels are running 0 0 eeeeeeeseeeeeeeeteeeeeeeeee 1 hour 7 Prepare second dimension running buffer 10 minutes Day 2 8 Disassemble tube apparatus eee cece cseceeeseceeeeees 2 minutes 9 Extrude gels from tubes and overlay tube gels onto slab Gel Secs sessa a need YR 25 minutes 10 Electrophorese the second dimension SDS gel 4 4 hours 10 2 Protocol for IEF First Dimension Casting IEF Tube Gels For reproducible 2 D gels it is essential that the IEF tube gels be precisely the same length and that polymerization be identical from day to day Care must be taken in pouring the gels to the same height so that the polymerization height will be the same from tube to tube An overlay step is not necessary in IEF first dimension tube gels The meniscus formed on top of the gel will not influence the pH gradient or the resolution of the bands The adva
23. coolant or water and capped 21 Section 9 Removing the Gels 1 After electrophoresis is complete turn off the power supply and disconnect the elec trical leads 2 Disconnect coolant hoses from the core if applicable and plug off the ports 3 Remove the cell lid and carefully pull the central cooling core out of the lower cham ber Pour off the upper buffer 4 Lay the central cooling core on its side and remove the sandwich assembly in the fol lowing manner With your index fingers below the sandwich clamps and your thumbs resting on the latch es in the cooling core gently remove the assembly by pulling up toward you in a man ner opposite to the way it was attached in Section 5 Pull the sandwich assembly off the locating pins on the top of the cooling core a 5 Loosen the single screw of each clamp and remove the clamps from the glass plates 6 Push one of the spacers of the sandwich out to the side of the plates without removing it 7 Gently twist the spacer so that the upper glass plate pulls away from the gel 22 8 Remove the gel by gently grasping two comers of the gel and lifting off or alternatively place the gel and glass plate under fixative solution and agitate gently until the gel separates from the glass plate 9 If the gel is to be stained later place it in a suitable container with fixative solution e g 40 methanol 10 acetic acid See Section 14 5 for staining formulation
24. dimensional technigues involve this variation of sample loading Either a cylin drical gel or a strip of a slab gel may be placed on top of a slab gel for separation into a sec ond dimension This procedure is described in detail in Section 10 Section 7 Running the Gel 1 Place thelid on top of the lower buffer chamber to enclose the PROTEAN II xi cell fully Note that the lid can be placed only in one orientation so that the anode and cathode con nections cannot be reversed 2 Attach the electrical leads to a suitable power supply such as those in Section 13 6 with the proper polarity this connection could accidently be reversed 3 Apply power to the PROTEAN II xi cell and begin electrophoresis As a safety pre caution always set voltage current and power limits when possible See the Appendix Section 14 4 for specific running conditions 19 Section 8 Set up Options 8 1 Buffer Recirculation Buffer recirculation is sometimes necessary for extended electrophoretic runs or for use with certain weak buffer systems To recirculate electrode buffer from the lower to the upper chambers a few simple modifications have to be made to the PROTEAN II xi cell 1 Locate the recirculation port tabs on the lid of the cell The tab directly above the upper buffer chamber and one of the tabs directly above the lower buffer chamber must be removed Remove by grasping with a pair of pliers and twisting the tab until it break
25. e with one dedicated instrument 1 2 Specifications Construction Cooling core and tube gel adaptor Lid and lower buffer chamber molded polycarbonate Clamps casting glass and Teflon filled molded poly stand and cams carbonate Electrical leads flexible coiled molded polysulfone Electrodes Shipping weight Overall size Gel size Glass plate sizes Cooling core maximum flow rate Maximum coolant temperature Voltage limit platinum 0 010 inch diameter 0 254 mm 11 kg 24 lb 3 oz 26 cm L x 19 cm W x 30 cm H 16 x 16 cm slab or 16 x 20 cm slab 1 to 6 mm diameter tube gels 16 cm cell 16x 20 cm inner plate 18 3 x 20 cm outer plate 2 liter min Do not exceed 50 C 1 000 volts DC Teflon is a registered trademark of E I DuPont de Nemours and Co Note PROTEAN II xi cell components are not compatible vvith ethanolamine ethylene diamine chlorinated hydrocarbons e g chloroform aromatic hydrocarbons e g toluene benzene or acetone Use of such organic solvents voids all warranties Cyanoacrylate and other adhesives will also attack the cell components Contact your local Bio Rad office for compatibility information before using any adhesive or organ ic solvent with this cell 1 3 Safety Power to the PROTEAN II xi cell and PROTEAN II xi 2 D cell is to be supplied by an external DC voltage power supply This power supply must be ground isolated in such a way that the DC voltage out
26. ecular weight range of 40 250 K Daltons Use in conjunction with Bio Rad High MW SDS PAGE Standards cata log number 161 0303 Calculated Volumes in ml Required Per Gel Slab Spacer Thickness 16 cm Length 20 cm Length 0 5 mm 12 8 ml 16 ml 0 75 mm 19 2 ml 24 ml 1 0 mm 25 6 ml 32 ml 1 5mm 38 4 ml 48 ml 3 0 mm 76 8 ml 96 ml Volume required to completely fill gel sandwich to top of plates Amounts may be adjusted depending on application with or without comb with or without stacking gel etc 40 14 3 Stacking Gel Preparation 4 0 gel 0 125 M Tris pH 6 8 16cm 20 cm Distilled water 6 1 ml 12 2 ml 0 5 M Tris HCl pH 6 8 2 5 ml 5 ml 10 w w SDS 100 ul 200 ul Acrylamide Bis 30 stock Degas for 15 minutes at room temperature 1 3 ml 2 6 ml 10 ammonium persulfate fresh daily 50 ul 100 ul 0590 TEMED 10 wl 20 ul 0 1 TOTAL STOCK MONOMER 10 ml 20 m Enough for two 0 75 mm or 0 50 mm gels Enough for two 1 5 or 1 0 mm gels 1 To prepare the monomer solutions combine all reagents except the APS and TEMED and deaerate under vacuum for gt 15 minutes To initiate polymerization add the APS and TEMED and swirl gently to mix 2 Follow the instructions in Sections 3 and 4 for set up and casting of the gels 14 4 Running Conditions We recommend that gels be run under constant current conditions with an appropriate power supply such as the PowerPac 3000 or Model 1000 500 power supply see Section 13
27. ell to leak If the pins are loose they can be gently tightened using a coin or screw driver 5 With your fingers below the latch on the cooling core and your thumbs resting on the clamps gently push the gel sandwich onto the cooling core with one simple motion The upper edge of the short inner glass plate should be butted against the notches of the U shaped gasket and the tabs of each clamp should be held securely against the latch assemblies on both sides of the cooling core 16 a Si D Turn the central cooling core to its other side and repeat steps through 5 to attach the second gel sandwich to it Note When the gel sandwich has been properly installed the shorter inside glass plate will be forced against the notch in the U shaped gasket to create a leak proof seal Always inspect the contact between the gasket and glass plate to make sure the glass plate is butted against the notch in the gasket and is not resting on top of or below this notch Improper installation of the gel sandwich can result in buffer leakage during the run As a standard procedure pour buffer into the upper buffer compartment and check for buffer leaks prior to a run In addition we recommend marking the level of the upper buffer on the glass plates prior to electrophoresis Checking this level after 1 2 hours will show whether a slow leak is occurring This is especially important when the elec trophoresis cell is being used for overnight experim
28. enter it Add APS and TEMED to the monomer solution and begin pumping the gradient As the monomer level in the sandwich approaches the tip of the needle withdraw the nee dle slowly so that it always stays above the monomer level Y i eh oi men Immediately rinse any remaining monomer out of the needle by pumping water through the gradient former and out the needle Overlay the gel or insert the comb as outlined in Sections 4 1 and 4 2 respectively 4 4 Casting Agarose Gels Agarose gels in vertical apparatus may slide down between the glass plates To prevent this one of the two plates used to form the agarose gel sandwich should be a frosted inner plate catalog number 165 1825 for 16 cm gels and catalog number 165 1826 for 20 cm gels 1 Assemble the glass plate sandwich as outlined in Section 3 using one frosted plate and one regular clear plate The frosted plate should be the shorter inner plate the plate on the inside during running This will allow visualization of the tracking dye during the run Make sure that the frosted side of the plate is on the inside of the gel sandwich Place the sandwich assembly in a warm air incubator 50 C for 5 10 minutes before casting the gel This will prevent premature gelling of the agarose Cam the warm assembly to the casting stand 13 4 Immediately pour the molten 55 to 60 C agarose One convenient method of pour ing is to tilt the sandwich as
29. ents 5 2 Use of the Buffer Dam If only one gel is to be run the acrylic buffer dam must be attached to the cooling core on the other side to form the complete upper buffer chamber Position the acrylic plate between two clamps by matching the notches on the clamps to the notches on the acrylic plate as in Section 3 1 steps 2 3 and then slide the dam up each clamp as far as possible No further alignment is necessary The acrylic buffer dam fits both 16 and 20 cm clamps 17 Note We do not recommend the use of the acrylic buffer dam at elevated temperatures as the block may warp and cause buffer leaks o Note Failure to slide the dam up completely to the top of the clamp will result in an upper buffer leak Section 6 Loading the Samples Sample loading can be done in one of three ways The most common method is to load liquid samples into wells formed by casting a gel with a well forming comb The second method uses the entire gel surface as a single well for liquid samples The third method involves placing a tube gel or gel strip over the entire gel surface as in 2 D procedures 6 1 Loading of Sample Wells The approximate sample volumes that each well will hold for all available combs is included in Section 13 2 1 2 Prepare 1 5 liters of electrode buffer Set aside 350 ml for the upper buffer chamber Pour 325 350 ml of electrode buffer into the upper buffer chamber At this point check the integri
30. first Pouring Gradient Gels from the Bottom 1 After assembling the gel sandwich as described in Section 4 attach the gradient former tubing to a gradient pouring needle catalog number 165 2007 2 Cam the gel sandwich to the casting stand turn the casting stand on its side locate the bottom filling ports directly under the space in the sandwich and insert the bottom fill needle through the rubber gasket so that it protrudes about 2 mm into the sandwich Make sure the needle opening faces one of the glass plates VS mg em em L 12 3 Stand the casting stand up on a level surface add APS and TEMED to the monomer solu tions and begin pumping the gradient monomer solution Once the gradient is poured this should take no more than 10 minutes from the time the initiators are added to the first monomer solution withdraw the needle from the gas ket and immediately clean the gradient former tubing and needle by pumping dis tilled water through them Overlay the monomer solution see Section 4 1 or insert the comb see Section 4 2 depending on whether the gel is discontinuous or continuous respectively Pouring Gradient Gels by Top Filling 1 Attach the gradient maker tubing to a needle catalog number 165 1943 a long can nula or a piece of tubing that will fit between the glass plates of the gel sandwich Insert the needle cannula or tubing as far as possible into the sandwich and c
31. g e Phone 01 810 16 77 e Fax 01 810 19 33 United Kingdom Bio Rad Laboratories Ltd Bio Rad House Maylands Avenue Hemel Hempstead Herts HP2 7TD e Phone 0800 181134 e Fax 0442 259118 Printed in USA M1651801 Rev B
32. he clamps the sandwich assembly is transferred to the alignment slot of the casting stand for final adjustments Section 3 Assembling the Glass Plate Sandwiches 3 1 Assembling Single Sandwiches Note Instructions for assembling 16 cm and 20 cm sandwiches are identical except of course for the lengths of the components To insure proper alignment make sure all plates and spacers are clean and dry before assembly The PROTEAN II xi plate wash er holder simplifies glass plate washing and also makes an ideal storage system for clean dry glass plates Each plate holder will accommodate up to 8 PROTEAN II xi plates or up to 18 Mini PROTEANS II plates 4 1 3 4 S Assemble the gel sandwich on a clean surface Lay the long rectangular plate down first then place two spacers of equal thickness along the long edges of the rectangular plate Next place a short plate on top of the spacers so that it is flush with one end of the long plate Locate both the right and left sandwich clamps and loosen the single screw of each by turning counterclockwise Place each clamp by the appropriate side of the glass plate stack with the locating arrows facing up and toward the glass plates Grasp the whole glass plate sandwich firmly with your right hand With your left hand guide the left clamp onto the sandwich so that the long and short plates fit the appro priate notches in the clamp Tighten the single screw enough to hold p
33. his makes 20 ml total volume enough to cast one set of gels using the casting tube Sample Solution A 1 0 g SDS 0 232 g DTT or DTE Dissolve in ddH20 to a final volume of 10 ml Store in aliquots at 70 C 42 Iso Urea Solution E 0 1 g DTT 0 4 g CHAPS 5 4 g urea 500 ul Bio Lyte 3 10 ampholyte 6 ml ddH2O Electrolytes First Dimension Upper Running Electrolyte Cathode 20 mM NaOH Dissolve 0 4 g NaOH in 500 ml deionized water and degas thoroughly for 30 min utes First Dimension Lower Running Electrolyte Anode 10 mM H3P04 Dilute 1 8 ml concentrated H PO in 2 6 liters deionized water and degas thoroughly for 30 minutes Transfer Solution 40 ml 0 5 M Tris HCl pH 8 8 80 ml 10 SDS 8 ml 0 05 bromophenol blue 150 ml ddH O 14 7 Running Conditions IEF is carried out at 200 volts constant voltage for 2 hours followed by 500 volts con stant voltage for 2 hours and finally 800 volts constant voltage for 16 hours overnight 43 Section 15 References 15 1 General References 1 Gordon A H Laboratory Techniques in Biochemistry and Molecular Biology Vol 1 Part 1 Work T S and Work E eds North Holland Publishing Co Amsterdam London 1975 Maurer H R Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis Walter de Gruyter Berlin New York 1971 15 2 Native Gel Systems References ON Ne LES Ritchie R F Harter J G and Bayles T B J Lab
34. issicisc ccsccesucsseatsbesssvedidbbs ese rine ares ree en Prr NE Sensini riti 12 Casting Gradient Gell oieri riere e edt E EEE EE 12 Casting Agarose Gels iiseisacsSiscsendssauccebssesse oia ek r Era atacant ies EIKER GR EERE 13 Assembling the Upper Buffer Chamber 15 E 15 Use of the Buffer Damien etat 17 Loading the Samples 18 L oadinsor sample Well u csscusvstesssenissansdstascsnsncoesvceasydoussdvesetysaoencseassdousssonncses 18 Loading a Single Sample Per Gels c csccscccsssscveesesscseesceesscgssecuasacsnavennsnsasssenesstionss 19 EE 19 Running the Gele ee tea meisi 19 Setup Options sikana div cause dessa ege DWN ISA ENEE SE EE pulun maa 20 Buffer Recrculaton Y EF 20 Cooling Opti0nS ee iie oss tasataan Te FFF d YN WCW EF GY Eeer 21 Removing the Gel 22 Two Dimensional Flectropboresis is naene 23 Sequence of Steps for 2 D Protocol eeueeiiesi iie di cyn ind GLWY Fn Fd Y 24 Protocol for IEF First D menston LY Y Y FFF FF FFF nnion 24 Maintenance of Kouipment AY A I IH YH Ynn Hon 30 Troubleshooting Guide PAGE SDS PAGE 2 D IEF SDS PAGE 31 Equipment and Accessories nnr nnr nn Ynn Ynn Anon 33 PROTEAN II xi Cell Slab Configurations een nne nenn nnsn nosi non 33 A GCGSSOF OSOD ee 33 PROTEAN II xi Cell 2 D Configuration nenn nr nni reies 36 ACCESSO OS nui ua yu YF Y i NU CYF GN HD 36 Electrophoresis ChemicalS centenes emet messi ens 31 13 6 Section 14 14 1 14 2 14 3 14 4 14 5 14 6 1
35. king gel is to be cast the following day place approximately 5 ml of 1 4 diluted stock solution B see Section 14 1 on top of each separating gel after rinsing with deionized water This will prevent dehydration of the separating gel during overnight storage at room tem perature Prepare the stacking gel monomer solution Combine all reagents except APS and TEMED and deaerate under vacuum at least 15 minutes Dry the area above the separating gel completely with filter paper before pouring the stacking gel 10 8 Place a comb in the gel sandwich and tilt it so that the teeth are at a slight 10 angle This will prevent air from being trapped under the comb teeth while pouring the monomer solutions 9 Add APS and TEMED to the degassed monomer solution and pour the solution down the spacer nearest the upturned side of the comb Pour until all the teeth have been covered by solution Then properly align the comb in the sandwich and add monomer to fill completely Generally an overlay solution is not necessary for polymerization when a comb is in place 10 Allow the gel to polymerize 30 45 minutes Remove the comb by pulling it straight up slowly and gently 11 Rinse the wells completely with distilled water The gels are now ready to be attached to the central cooling core the sample loaded and the gels run 11 4 2 Casting Continuous Gels Continuous gels are ones in which the entre gel is of one composition This ty
36. l 2 D electrophoresis native electrophoresis and agarose gel electrophoresis The PROTEAN II xi cell can run up to 4 slab gels or 16 tube gels simultaneously The basic unit accommodates gels 16 or 20 cm long The 20 cm gels offer increased resolution capability which is especially useful in 2 D applications The central cooling core of the PROTEAN II xi cell assures even heat distribution over the entire gel length permitting excellent resolution with minimal band distortion Only 1 5 liters of buffer are required The raised electrode position insures safe operation even with extended overnight runs The unique single screw sandwich clamps allow rapid assembly of the gel sandwich es while providing even pressure distribution along the entire gel length This even pres sure minimizes the risk of breaking the glass plates a common problem with multi screw clamps The PROTEAN II xi alignment card helps keep spacers upright during sandwich alignment The combination of the clamps alignment card and the casting stand permits assembly and casting of gels in minutes with little effort After casting the completed gel sandwich attaches to the central cooling core with a single motion The PROTEAN II xi cell is the instrument of choice for 2 D electrophoresis With the optional tube adaptors one can run the first dimension IEF tube gel and then overlay this gel onto the second dimension SDS slab gel Thus the complete 2 D procedure can be don
37. lamps consists of a left clamp and a right clamp The sandwich clamps can accommodate up to two 1 5 mm thick gels 2 3 Casting Stand The casting stand is separate from the PROTEAN II xi cell so that two gel sandwich es can be cast while others are being run The one piece molded unit has two casting slots and one alignment slot Gel sandwiches are assembled aligned and cammed into the stand quickly without the use of grease Double gels may also be cast Two 1 5 mm or thinner gels may be cast in each sand wich so that up to four 1 5 mm gels can be run at once 2 4 Upper Butter Chamber The completed gel sandwich attaches to the central cooling core so that the outer plate of the sandwich forms the side of the upper buffer chamber The inner plate is clamped against a rubber gasket on the central cooling core to provide a greaseless leak free seal for the upper buffer Each sandwich forms one side of the cathode chamber Tube gel adaptors also snap onto the central cooling core to form the upper buffer chamber walls one adap tor per side If only one gel is to be run an upper buffer dam is attached to the core to form the complete upper buffer chamber The upper buffer chamber will hold approxi mately 350 ml of buffer when full 2 5 Lower Buffer Chamber The lower buffer chamber of the PROTEAN II xi cell encloses the unit and provides sta bility during electrophoresis The molded unit requires a minimum buffer volume of only 1 1
38. lates in place Place the right clamp on the right side of the plates and tighten the clamp screw Level the casting stand on a flat surface with the alignment slot facing you Check to see that gaskets are clean and free of residual acrylamide to insure a good seal Place a flat grey silicone gasket in each casting slot Note Always use the alignment slot to properly orient the gel sandwich Failure to use this slot for alignment can result in casting leaks while pouring the gel or buffer leaks during the run 6 Place the assembled gel sandwich in the alignment slot of the casting stand Loosen the clamp screws and allow the plates and spacers to align at the surface of the alignment slot Insert a PROTEAN II xi alignment card between the two glass plates to keep the spacers upright while additional alignment adjustments are made As an alternative the alignment card can be positioned between the glass plates when the sandwich is first assembled as in step 1 Ss 7 Simultaneously push inward on both clamps at the locating arrows and tighten both clamp screws just enough to hold the sandwich in place Pushing inward on both clamps at a point below the locating arrows will insure that the spacers and glass plates are flush against the sides of the clamps 8 Loosen one clamp screw slightly While pushing down on the spacer with one finger tighten the clamp screw finger tight with the other hand see photo This will insure prop
39. locator at the bottom of the unit to align the tubes Plug any unused tube holes with a stopper Prepare the sample just before loading The amount of denaturing sample solution A and or iso urea solution E will depend upon the protein concentration of sample and upon the type of sample An initial starting ratio of 1 ul IEF sample concentrate for every 10 ul sample can be used For denaturing samples are heated at 95 9C for 5 minutes then cooled for 2 minutes at room temperature before loading or before adding iso urea solution Load the samples with a Hamilton syringe or with a Drummond pipet tip Generally 15 to 30 ul of final diluted sample is loaded 26 8 Carefully overlay the sample with upper electrolyte 20 mM NaOH Running the IEF Gels 1 Fill the central cooling core with water or coolant Cap the inlet and outlet port with the caps provided Fill the lower buffer chamber with 4 5 liters of lower running electrolyte see Section 14 Place on a level surface or leveling table Lower the cooling core tube apparatus into the lower chamber of the PROTEAN II xi cell Lower carefully so as not to introduce any air bubbles under the gel tubes All bubbles must be removed from the bottom of the tubes to insure proper electrical con tact Pour 325 350 ml freshly degassed upper running electrolyte see Section 14 into the upper buffer chamber put the lid on and attach the power cables to the power supply Run the
40. m spacer 4 15 well comb 2 165 1812 1 0 mm spacer 4 15 well comb 2 165 1813 0 75 mm spacer 4 15 well comb 2 165 1814 All PROTEAN II xi cells include the central cooling core with gaskets and core caps lower buffer chamber lid with power cables 2 sets of glass plates 4 sandwich clamps 4 spacers 2 combs an upper buffer dam a casting stand with gaskets a leveling bubble alignment card and instructions Cells contain all of the above except spacers and combs order separately 13 2 Accessories Catalog Number Product Description 16 cm cell 20 cm cell Glass Plates Inner Plate 2 165 1821 165 1823 Outer Plate 2 165 1822 165 1824 Frosted Inner Plate 2 agarose gels 165 1825 165 1826 Beveled Inner Plate 2 2 D procedures 165 1827 165 1828 Notched Inner Plate 1 double up 165 1832 165 1833 procedures Note One complete gel sandwich consists of 1 outer long and 2 spacers plate 1 inner short plate One complete double up sandwich 2 gels side consists of 1 outer plate 1 inner plate 1 notched inner plate and 4 spacers See Section 3 2 for assembly Used in conjunction with regular outer plate PROTEAN II Plate Washer Holder Plate Washer System includes Plate Holders 2 165 1991 Tank and Lid Plate Holder 1 165 1992 33 Product Description Catalog Number 16 cm cell 20 cm cell Spacers set of 4 0 5 mm 0 75 mm 1 0 mm 1 5 mm 3 0 mm Sandwich Clamps
41. ms References ED EN ca aged TA O Farrell P H J Biol Chem 250 4007 1975 Ferro Luzzi Ames G and Nikaido K Biochem 15 616 1976 Anderson L and Anderson N G Proc Nat Acad Sci U S A 74 5421 1977 O Farrell P Z Goodman H M and O Farrell P H Cell 12 1133 1977 Garrels J I J Biol Chem 254 7961 1979 Hochstrasser D F Harrington M G Hochstrasser A C Miller M J and Merril C R Analytical Biochemistry 173 424 435 1988 Hochstrasser D F Augsburger V Funk M Appel R Pelegrini C and Muller A F Electrophoresis 7 505 511 1986 44 Life Science Group 2000 Alfred Nobel Drive Hercules California 94547 Telephone 510 741 1000 Fax 510 741 1060 Bio Rad Laboratories Eastern Regional Office 85A Marcus Dr Melville New York 11747 e Phone 516 756 2575 e Fax 516 756 2594 European Headquarters Bio Rad Laboratories Dreve du S n chal 19 B 1180 Brussels e Phone 02 375 59 70 e Fax 02 374 61 62 Australia Bio Rad Laboratories Pty Limited Unit 11 112 118 Talavera Rd P O Box 371 North Ryde N S W 2113 e Phone 02 805 5000 e Fax 02 805 1920 Austria Bio Rad Laboratories Ges m b H Auhofstrasse 78D A 1130 Wien e Phone 0222 877 89 01 e Fax 0222 876 56 29 Belgium Bio Rad Laboratories S A N V Begoniastraat 5 B 9810 Nazareth Eke e Phone 091 85 55 11 e Fax 091 85 65 54 Canada Bio Rad Laboratories Canada Ltd 5149 Bradco Boulevard Mi
42. nd leveling bubble alignment card and instructions 13 4 Accessories Catalog Product Description Number Tube Gel Adaptor with a complete set of grommets 165 1940 4 8 mm O D tubes and stoppers Tube Gel Adaptor Replacement Gaskets 2 165 1947 Stoppers 8 165 1941 Grommets and Stoppers for 4 5 mm OD Tubes 12 165 1984 Grommets and Stoppers for 6 7 mm OD Tubes 12 165 1985 Gel Tube Loading Needle 18 cm 22 gauge blunt tip 165 1943 Luer hub for casting monomer in small diameter tubes Gel Tube Extrusion Needle 9 cm 26 gauge beveled 165 1944 tip Luer hub for removing gels from tubes PROTEAN II xi Multi Gel Casting Chamber 165 2025 36 13 5 Electrophoresis Chemicals Quantity per Catalog Product Description Package Number Acrylamide 99 9 100 g 161 0100 Acrylamide 99 9 500 g 161 0101 Acrylamide 99 9 1 kg 161 0107 Acrylamide 99 9 2kg 161 0103 Preweighed Acrylamide Bis 30 g 161 0122 37 5 1 mixture Preweighed Acrylamide Bis 150 g 161 0125 37 5 1 mixture Bis N N Methylene bis acrylamide 5g 161 0200 Bis N N Methylene bis acrylamide 50 g 161 0201 Piperazine di Acrylamide PDA 10 g 161 0202 Tris 500 g 161 0716 Tris 1 kg 161 0719 Glycine 250 g 161 0717 Glycine 1 kg 161 0718 Boric Acid 500 g 161 0750 Boric Acid 1 kg 161 0751 SDS Sodium dodecylsulfate 258 161 0300 SDS Sodium dodecylsulfate 100 g 161 0301 SDS Sodium dodecylsulfate 1kg 161 0302 Ammonium Persulfate 10 g 161 0700 Dithiothreitol lg 1
43. nner glass plates and a set of four spacers of equal length 1 Lay down a long rectangular plate two spacers and a notched inner plate 2 Place two more spacers on top of the notched inner plate Place the short inner glass plate on top of this set of spacers to form the complete dou ble sandwich 3 Apply the sandwich clamps as described in Section 3 1 steps 3 4 Insert two align ment cards between each of the sandwiches to keep the spacers upright during sandwich alignment Align and then cam the whole assembly into the casting stand The sand wiches are now ready for gel casting Section 4 Casting the Gels 4 1 Casting Discontinuous Laemmli Gels Discontinuous gels consist of a resolving or separating lower gel and a stacking upper gel The stacking gel acts to concentrate large sample volumes resulting in better band resolution than is possible using the same volumes on a gel without a stack Molecules are then completely separated in the resolving gel The most popular discontinuous buffer system is that of Laemmli This formulation is included in the Appendix Laemmli U K Nature 227 680 1970 1 Prepare the monomer solution by combining all reagents except ammonium persulfate APS and TEMED see Section 14 1 for formulations Deaerate the solution under vac uum for at least 15 minutes 4 Place a comb completely into the assembled gel sandwich With a marker pen place a mark on the glass plate 1
44. nt by 25 50 9 Run too fast poor resolu a Buffer too dilute a Check buffer protocol tion dilute if necessary b Current too high b Decrease current by 25 50 10 Doublets observed where a A portion of the protein a Prepare fresh sample buffer a single protein species is may have been re oxidized solutions if over 30 days expected SDS PAGE during the run or may not old increase B mercap have been fully reduced toethanol concentration in prior to run the sample buffer 11 Observe fewer bands a More than one band migrat a Increase T of resolving than expected and one ing at the dye front gel heavy band at dye front 12 Nonlinear pH gradient at a Upper electrolyte depleted a Increase the concentration of basic end upper electrolyte to 100 mM Polyacrylamide gels are described by reference to two characteristics 1 The total monomer concentration T 2 The crosslinking monomer concentration C YT C gm Acrylamide gm Bis Acrylamide Total Volume gm Bis Acrylamide x 100 x 100 gm Acrylamide gm Bis Acrylamide 32 Section 13 Equipment and Accessories 13 1 PROTEAN II xi Cell Configurations Catalog Product Description Number PROTEAN II xi Cells PROTEAN II xi 16 cm Cell 165 1801 1 5 mm spacer 4 15 well comb 2 165 1802 1 0 mm spacer 4 15 well comb 2 165 1803 0 75 mm spacer 4 15 well comb 2 165 1804 PROTEAN II xi 20 cm Cell 165 1811 1 5 m
45. ntage of not overlaying is the formation of gels of more uniform length and composition Stock solutions and formulations for first dimension tube gels are given in Section 14 1 Mark the capillary tubes 1 5 mm ID 6 0 mm OD 180 mm catalog number 165 3138 with a laboratory marker 14 0 cm from one end 2 Connect each capillary tube to a 1 ml syringe using a small piece of Tygon tubing 316 ID x 1A OD and approximately 2 cm in length not included Fill either a test tube rack or a level casting stand such as Bio Rad s Model 225 Tube Gel Casting Stand cat alog number 165 2020 with a disposable 12 x 75 mm test tube for each capillary tube Insert a capillary tube syringe assembly into each test tube 3 Prepare the first dimension monomer solution and degas well The removal of molec ular oxygen by degassing is essential for reproducible polymerization Warning Always wear gloves to prevent exposure to acrylamide 4 Add the APS and TEMED and swirl 8 to 10 times Working quickly pipet 1 ml of acry lamide solution into each test tube Using the syringe pull up the liquid in each tube to the 14 0 cm mark Let the capillary tubes sit undisturbed with syringes attached for 2 hours at room temperature to allow complete polymerization to occur 24 After polymerization is completed remove the capillary tubes from each test tube Remove the syringe and Tygon tubing Press and rotate the bottom of the capillary tube squ
46. olution scrub out if possible then rinse with distilled H O Store glass tubes in chromic sulfuric acid solution until next use Then rinse thoroughly with distilled water and dry in forced air or vacuum oven before use Warning Exercise extreme caution for acid cleaning wear safety glasses a lab coat and rubber gloves Keep a container of NaCO nearby to neutralize spills 30 Section 12 Troubleshooting Guide PAGE SDS PAGE 2 D IEF SDS PAGE Problem Cause Solution 1 Smile effect band pattern curves upward at both sides of the gel 2 Diffuse tracking dye 3 Vertical streaking of protein 4 Horizontal streaking 2 D gels 5 Broad or diffuse protein bands or spots 2 D 6 Lateral band spreading Center of the gel running hotter than either side Power conditions exces sive Decomposition of sample solution and or buffer stock solutions Diffusion Sample overload Sample precipitation Incomplete solubilization prior to first dimension Interfering nucleic acids in sample Diffusion due to slow migration Chemical changes due to ionic contaminants in urea Diffusion out of the wells prior to turning on the cur rent Diffusion during migration through the stacking gel 31 Fill inner core with coolant Circulate coolant at 10 15 C Decrease power setting Prepare fresh reagents maximum shelf life of aqueous solutions is 30 days at
47. pe of gel is often used in non denaturing native buffer systems 1 Prepare the monomer solution Combine all reagents except APS and TEMED Degas under vacuum for at least 15 minutes 2 Place a comb in the glass sandwich so that the teeth are tilted at approximately a 10 angle 3 Add APS and TEMED to the degassed monomer solution and use a pipet and bulb to pour the solution down the spacer nearest to the upturned side of the comb Pour until the bottoms of all the teeth are covered Then adjust the comb to its proper position Add monomer solution to fill the sandwich completely No overlay solution is necessary 4 Let the gel polymerize for 45 minutes to 1 hour The gel is now ready to load and run Remove the comb and rinse thoroughly with distilled water 4 3 Casting Gradient Gels Polyacrylamide concentration gradients made with an external gradient former Model 385 catalog number 165 2000 can be introduced into the PROTEAN II xi gel sandwich assembly either from the bottom or from the top A peristaltic pump is required for intro duction from the bottom Introduction from the top can be done by gravity flow or with a peristaltic pump Follow the gradient former instructions for formulating the gradient If gra dients are pumped into the gel sandwich from the bottom the low density solution low per cent monomer must enter first If the gradient enters from the top the high density solution high percent monomer enters
48. pecifically designed for use during the first dimension of 2 D electrophoresis Do not lubricate or wet the red tube adaptor gaskets Note Tube gels have a much higher resistance than slab gels due to their small diam eter Since current seeks the path of least resistance a current leak may occur if there is an alternative path of conductance such as a wet gasket A current leak is a safety haz ard to the researcher as well as the equipment Attach the tube gel adaptor to the cooling core in the same manner that a slab gel sand wich is attached see Section 5 1 Sandwich clamps are not needed to attach the tube adaptor to the core Finish assembling the upper buffer chamber with a second tube gel adaptor Because of the higher voltages required for focusing we recommend always using two tube gel adaptors and not the buffer dam for focusing 25 6 7 Note Buffer leakage during isoelectric focusing can result in damage to the cooling core It is important to check for buffer leaks by monitoring both the current and the upper buffer level During the course of a normal IEF run the current decreases as the resis tance of the gel increases the pH gradient is established and the upper buffer level is maintained If a buffer leak should occur the current will increase and the level of upper buffer may decrease Do not exceed 1 000 V as the maximum focusing voltage Insert the tube gels into the tube gel adaptor using the gel tube
49. put floats with respect to ground All of Bio Rad s power supplies meet this important safety requirement Regardless of which power supply is used the maximum specified operating parameters for these cells are 1000 VDC maximum voltage limit 80 Watts maximum power limit 50 C maximum ambient temperature limit Current to the cell provided from the external power supply enters the unit through the lid assembly providing a safety interlock to the user Current to the cell is broken when the lid is removed Do not attempt to circumvent this safety interlock and always turn the power supply off before removing the lid or when working with the cell in any way Important This Bio Rad instrument is designed and certified to meet IEC1010 1 safety standards Certified products are safe to use when operated in accordance with the instruction manu al This instrument should not be modified or altered in any way Alteration of this instru ment will e Void the manufacturer s warranty e Void the IEC1010 1 safety certification e Create a potential safety hazard Bio Rad is not responsible for any injury or damage caused by the use of this instrument for purposes other than for which it is intended or by modifications of the instrument not per formed by Bio Rad or an authorized agent IEC 1010 1 is an internationally accepted electrical safety standard for laboratory instruments Section 2 Description of Major Parts Fig 2 1 PROTE
50. s If the proteins on the gel are to be electrophoretically transferred to a membrane place the gel in equilibration buffer do not put in fixative Note The Model 556 Gel Destainer catalog number 165 2010 is ideal for rapid destaining less than hour of Coomassie blue stained gels Section 10 Two Dimensional Electrophoresis Two dimensional electrophoresis can provide exceptionally high resolution of the pro tein components in a complex sample It is capable of resolving several thousand individ ual protein species Based on the method of O Farrell the first dimension is isoelectric focusing IEF during which proteins are separated according to their isoelectric points The second dimension is SDS polyacrylamide gel electrophoresis in which proteins are sepa rated on the basis of their molecular size Since O Farrell s original work many variations of the 2 D procedure have been reported which may also be used The following procedure is based on the work of Dr Denis Hochstrasser The flow chart Section 10 1 outlines in sequence the essential steps of 2 D electrophoresis and refers to the solution protocols in Section 14 Note This section focuses on 2 D electrophoresis The PROTEAN II xi cell may also be used for one dimensional tube gel electrophoresis One can adapt this protocol to any of the common electrophoretic techniques using either continuous or discontinuous buffer systems by following the instructions for
51. s off see photo Locate the punch out tab in the upper right corner on both sides of the central cooling core Carefully bore one of the thin membrane tabs with a drill and 1 4 in 6 mm bit Insert a section of tubing down through the port above the lower buffer chamber and to the bottom of the lower buffer chamber Run this tubing through a peristaltic pump and into the upper buffer chamber through its port Buffer is then pumped from the lower to the upper buffer chamber As the upper cham ber fills buffer will overflow through the port on the core back into the lower buffer cham ber The buffer flowing through the tubing and pump is electrically active For this reason handle the tubing carefully when the power supply is on Do not touch any exposed liq uid with the power supply on Tube connections should be made with the power sup ply turned off Both the recirculation pump and the recommended power supplies must be ground isolated by design to minimize the potential for shock hazard However working around high voltage equipment in a laboratory environment is potentially dan gerous As a result it is the user s responsibility to always exercise care in setting up and running electrophoresis instruments If a liquid leak occurs always turn off the power supply before correcting the problem 20 Note Recirculation can only be used with continuous buffer systems i e systems in which the anode buffer and cathode b
52. s Bio Rad Cleaning Concentrate catalog number 161 0722 and rinsed thoroughly with distilled water before use Model Catalog No Date of Delivery Warranty Period Serial No Warranty Bio Rad Laboratories warrants the PROTEAN II xi cell against defects in materials and workmanship for 1 year If any defects occur in the instrument during this warranty peri od Bio Rad Laboratories will repair or replace the defective parts free The following defects however are specifically excluded Defects caused by improper operation NO Repair or modification done by anyone other than Bio Rad Laboratories or an autho rized agent 3 Use of fittings or other spare parts supplied by anyone other than Bio Rad Laboratories 4 Damage caused by accident or misuse 5 Damage caused by disaster 6 Corrosion due to use of improper solvent or sample This warranty does not apply to parts listed below 1 Platinum wire 2 Glass plates For any inquiry or request for repair service contact Bio Rad Laboratories after con firming the model and serial number of your instrument Section 1 General Information 1 1 Introduction The PROTEAN II xi cell is a vertical slab electrophoresis instrument which combines versatility with practicality When used with the various combs spacers and accessories avail able the PROTEAN II xi cell is suitable for most common electrophoretic techniques including SDS electrophoresis two dimensiona
53. sembly and pour agarose directly down the long rectangu lar glass plate Note If an incubator is not available molten 75 to 85 C agarose can be poured into a room temperature sandwich assembly This temperature is high enough to prevent pre mature gelling of the agarose yet low enough to prevent cracking of the glass plates Insert the prewarmed comb carefully Allow the agarose to cool to ambient temperature before use Remove the comb very slowly to avoid tearing the gel Note There are some special tricks to properly remove a comb from a vertical agarose gel The most important point is to introduce water or buffer under the comb while it is being removed To introduce buffer or water use a squirt bottle or a needle and syringe to force fluid under the teeth of the comb while it is slowly removed from the gel Another option is to insert the comb only partway into the gel This can easily be done with the aid of comb conversion screws catalog number 165 1859 The three standard screws on the comb are replaced with the three large head comb conversion screws with the protruding head of the conversion screws resting on top of the longer outer glass plate The well depth of the comb is limited to 10 mm instead of the stan dard 25 mm well depth 14 A Comb conversion screw B Standard comb screw Section 5 Assembling the Upper Buffer Chamber 5 1 Assembly 1 Lay the central cooling core down flat on a lab bench
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55. ty of the upper buffer seal If the buffer appears to be leaking remove the gel sandwich assemblies re lubricate the gasket and then re attach the gel sandwich assemblies as in Section 5 Place the remainder of the electrode buffer into the lower buffer chamber Lower the cen tral cooling core into the lower buffer chamber at a slight angle to prevent air entrapment under the gel sandwich A few isolated bubbles under the gel will not affect the run With 16 cm plates it is necessary to dilute the lower buffer with distilled water to a level of 1 cm above the bottom of the gel plates Be sure to mix the lower buffer well with a stir bar on a magnetic stirrer Note Dilution of the lower buffer by up to 1 2 with dH O will have no detrimental effect on resolution Dilution of the upper buffer is not recommended Load the samples into the wells under the electrode buffer with a Hamilton syringe Insert the syringe to about 1 2 mm from the well bottom before delivery Disposable pieces of plastic tubing may be attached to the syringe to eliminate the need for rinsing the syringe between samples 18 Note The sample buffer must contain either 1090 sucrose or glycerol in order to under lay the sample in the vvell vvithout mixing 5 An easier method of sample loading is to use an Eppendorf like pipettor and disposable tips To accomplish this successfully use the optional beveled short plate catalog num ber 165 1827 for 16 cm and 165 1828
56. uffer are the same 8 2 Cooling Options The cooling core may be used in any of the following ways 1 The core can be connected to any circulating cooler Any common anti freeze or ethy lene glycol 20 80 may be circulated through the core Do not use ethanol or methanol for coolant For most SDS PAGE and 2 D applications temperature should be set between 10 and 15 C For some specialized applications employing native or non denaturing gel systems in which temperature sensitive enzymes or other labile pro teins are to be separated the system can be cooled to 2 4 C For these applications greatest heat transfer efficiency is achieved by circulating coolant through the core at 2 C and filling the lower buffer chamber to the top of the slab gels The lower buffer is continually circulated by stirring 2 The core may be filled with coolant and the circulation ports plugged off with the core caps The coolant will act as a heat sink during electrophoresis This option works espe cially well for SDS PAGE 3 Distilled tap water may be circulated through the core 4 Temperature of the coolant can be monitored by placing a thermometer through one of the ports in the lid of the PROTEAN II xi cell The temperature of the upper and lower buffers will equilibrate to the temperature of the coolant in about 1 2 hour Remove the tab in the lid as in Section 8 1 step 1 5 During periods of non use the cooling core can be left filled with
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