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Western Blotting Handbook and Troubleshooting Guide Version 2

1. STEP STEP SDS PAGE Electro Tra r The choice of a primary antibody for a Western blot will depend on the antigen to be detected and what antibodies are available to that antigen A huge number of primary antibodies are available commercially and can be identified quickly by searching sites such as www antibodyresource com or www linscottsdirectory com Alternatively a primary antibody may be made to recognize the antigen of interest Both polyclonal and monoclonal antibodies work well for Western blotting Polyclonal antibodies are less expensive and less time consuming to produce and they often have a high affinity for the antigen Monoclonal antibodies are valued for their specificity purity and consistency that result in lower background Crude antibody preparations such as serum or ascites fluid are sometimes used for Western blotting but the impurities present may increase background To obtain antibodies with the greatest specificity they can be affinity purified using the immobilized antigen For more information on affinity purification request your FREE Affinity Purification Handbook from our website or contact a customer service representative at 800 874 3723 or 815 968 0747 Outside the United States contact your local distributor A wide variety of labeled secondary antibodies can be used for Western blot detection The choice of secondary antibody depends upon the species of animal in which the primary anti body was
2. 2 3 4 Mouse anti Human 1 500 1 500 1 1 000 1 1 000 p53 1 ug ml 1 ug ml 0 5 ug ml 0 5 pg ml Goat anti Mouse 1 1 000 1 5 000 1 10 000 1 20 000 HRP 1 pg ml 0 2 ug ml 0 1 pg ml 0 05 ug ml Exposure Time 30 seconds 30 seconds 1 minute 1 minute Figure 2 Example of signal intensity on a Western blot when using Thermo Scientific SuperSignal West Pico Substrate and antibodies at various concentrations Recombinant Human Wild Type p53 Baculovirus lysate at various concentrations was electrophoretically separated and transferred to nitrocellulose membrane The membrane was blocked with BSA and then incubated with various dilutions of mouse anti human p53 starting at the manufacturer s recommended dilution HRP labeled goat anti mouse was added at different concentrations and the signal was developed with SuperSignal West Pico Substrate The exposure times were also varied as indicated In Blot 1 the blot was totally black due to both the primary and secondary antibody concentrations being too high In Blot 2 the background is inconsistent but very dark again a result of too much primary and secondary antibody In Blots 3 and 4 the signal to noise was much better because both the primary and secondary antibody concentrations were reduced Neither blot 3 nor 4 had background signal For more information or to download product instructions visit www thermo com pierce Enzyme Substrates funooysajqnosy Primary Antibo
3. Remove the blot from the substrate working solution and place itin a plastic membrane protector A plastic sheet protector works very well although plastic wrap may also be used Remove all air bubbles between the blot and the surface of the membrane protector Place the wetted blot against the film and expose Standard autoradiographic film can be used A recommended first exposure time is 60 seconds Vary exposure time to obtain optimum results The use of enhanced or pre flashed autoradiographic film is unnecessary Note If a cooled CCD Camera e g Alpha Innotech Corporation s Chemilmager Camera is used longer exposure times may be necessary 14 Develop the film using appropriate developing solution and fixative for the type of film used 15 On an optimized blot the light generated should last a minimum of six hours The blot can be re exposed to film as needed to obtain the optimal results Longer exposure times may be necessary as the blot ages funooysajqnosy Bers G and Garfin D 1985 Protein and nucleic acid blotting and immunobiochemical detection BioTechniques 3 276 288 Bjerrum 0 J and Heegaard N H H 1988 Handbook of Immunoblotting of Proteins Volume 1 Technical Descriptions CRC Press Boca Raton Bollag D M et a 1996 Protein Methods Second Edition Wiley Liss Inc New York Product 20001 Gallagher S 1996 Immunoblot Detection Current Protoco
4. Formul Wash Buff Block the nonspecific sites on the membranes by incubating them in blocking buffer that contains 0 05 Tween 20 blocker Tween 20 Detergent for 1 hour at RT with shaking Prepare the primary antibody dilutions in blocker Tween 20 Detergent and apply to the membranes Incubate for 1 hour at RT with shaking Recommended Primary Antibody Dilutions from 1 mg ml stock 1 100 1 5 000 or 0 2 10 ug ml Thermo Scientific Pierce Substrate Pierce ECL Substrate SuperSignal West Pico Substrate 1 1 000 1 5 000 or 0 2 1 0 ug ml SuperSignal 1 5 000 1 100 000 or 0 01 0 2 ug ml West Femto Substrate SuperSignal 1 1 000 1 50 000 or 0 02 1 0 ug ml West Dura Substrate Lumi Phos WB Substrate 1 200 1 2 000 or 0 5 5 0 ug ml Wash the membrane four to six times in TBS or PBS using as large a volume of wash buffer as possible Add 0 05 Tween 20 Detergent to the wash buffer to help reduce nonspecific back ground For each wash suspend the membrane in wash buffer and agitate for approximately 5 minutes Pour off the wash buffer and repeat Brief rinses of the membranes before incubation in the wash buffer may increase the wash step efficiency Prepare dilutions of the secondary antibody HRP conjugate in blocker Tween 20 Detergent Add the secondary antibody dilutions to the membranes and incubate for 1 hour with shaking Recommended Secondary Antibody Dilutions from 1 mg ml stock 1 1 000 1 15 000 or 0 067 1 ug
5. 88585 88518 Low Fluorescence PVDF Transfer Membrane 0 2 pm 7 cm x 8 4 cm PVDF Transfer Membrane 0 45 pm 10 cm x 10cm PVDF Transfer Membrane 0 45 pm 26 5 cm x 3 75 m Western Blotting Filter Paper Product Description 88600 Western Blotting Filter Paper For more information or to download product instructions visit www thermo com pierce Pkg Size 15 pkg 1 roll 15 pkg 15 pkg 25 pkg 15 pkg 25 pkg Pkg Size 10 pkg 10 sheets 1 roll Pkg Size 100 sheets Enzyme Substrates Thermo Scientific Pierce Reversible Protein Stain for Nitrocellulose and PVDF Membranes A great alternative to Ponceau S stain For years the red Ponceau S has been the best option for staining before Western blotting despite its major shortcomings Pierce Reversible Protein Stains decrease staining time increase staining sensitivity and enhance the immunoreactivity of antigens in subsequent Western blotting Figures 2 4 Try these reversible protein stains for nitrocellulose and PVDF membranes and you will never use Ponceau S again Highlights e Sensitive general protein stain that binds tightly to proteins e Stain is protein specific avoiding interference from other biomolecules e From stain to destain in minutes e Turquoise bands are easily photographed e Stained bands do not fade with time e Enhances Western blot detection e All components are room temperature stable Figure 2 T
6. Electro Transfer Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate Twice as much signal for about 40 less than the price of the GE Healthcare Amersham ECL System In side by side comparisons using identical conditions blots incubated in SuperSignal West Pico Chemiluminescent Substrate exhibit at least twice the intensity of blots treated with the GE Healthcare Amersham ECL System More stable SuperSignal West Pico Substrate is room temperature RT stable for months with no discernable loss in activity RT stability frees up valuable cold room space and saves time because there is no need to wait for the reagents to warm up Long signal With signal duration of more than six hours there is adequate time to optimize the exposure conditions In most cases there is no need to rerun samples and repeat the blotting procedure STEP STEP Formul Blocki Wash Buff Highlights e Economy costs less per ml than other chemiluminescent substrates Table 2 e Long light emission strong light emission over a working day allows you to make several exposures e High intensity signal is twice as intense as other compatibly priced luminol based systems Figure 3 e Picogram sensitivity highly sensitive for the rapid development of a wide range of protein levels Figure 4 e Excellent stability 24 hour plus working solution stability kit is stable for at least one year at room temperat
7. Western Blotting Handbook and Troubleshooting Guide Featuring Thermo Scientific SuperSignal Substrates and Pierce Western Blotting Accessories Ti BB Version 2 Part of Thermo Fisher Scientific Introduction Western Blotting Overview Step 1 SDS PAGE Thermo Scientific Precise Protein Gels Molecular Weight Markers Step 2 Electro Transfer Thermo Scientific Pierce Fast Transfer System Transfer Buffers Filter Paper for Blotting PVDF and Nitrocellulose Membranes Thermo Scientific Pierce Protein Stains for Membranes Antibody Extender Solution NC Western Blot Signal Enhancer Step 3 Blocking Introduction Blocking of Nonspecific Binding Sites on Transfer Membranes Blocking Buffer Optimization Blocking Buffers Step 4 Formulate Wash Buffers Washing the Membrane Wash Buffers Step 5 Detection Reagents Validated Primary Antibodies Affinity purified Antibodies Stabilized HRP Conjugates Thermo Scientific DyLight Fluor Conjugates Conjugate Stabilizer Solutions Thermo Scientific DyLight labeled Highly Cross Adsorbed Secondary Antibodies Antibody Storage and Stabilizer Solutions Secondary Antibody Ordering Table Thermo Scientific Clean Blot IP Detection Reagents Protein A G A G and L Conjugates Thermo Scientific NeutrAvidin Streptavidin and Avidin Conjugates MO D o o N 12 12 1213 13 16 17 17 18 19 20 21 22 22 23 24 25 26 25 29 31 33 Step 6 Enzyme Substrate
8. e Affinity chromatography e Fluorescent activated cell sorting FACS Comparison of Thermo Scientific NeutrAvidin Biotin Binding Protein Avidin and Streptavidin Protein MW pl Thermo Scientific NeutrAvidin Biotin Binding Protein Thermo Scientific Streptavidin Thermo Scientific Avidin Carbohydrate 60 kDa 6 3 No 53 kDa 6 8 7 5 No 67 kDa 10 Yes Thermo Scientific NeutrAvidin Products For ultralow nonspecific binding compared to avidin or streptavidin Achieve better assay results with the low nonspecific binding properties of NeutrAvidin Protein NeutrAvidin Biotin Binding Protein is a deglycosylated form of avidin so lectin binding is reduced to undetectable levels without losing biotin binding affinity K 10 M NeutrAvidin Biotin Binding Protein offers the advantage of a neutral pl to minimize nonspecific adsorption along with lysine residues that remain available for derivatization or conjugation through amine reactive chemistries The molecular weight of NeutrAvidin Biotin Binding Protein is approximately 60K The specific activity for biotin binding is approximately 14 ug mg of protein which is near the theoretical maximum activity Highlights e Near neutral pl 6 3 and no glycosylation unlike avidin e No RYD recognition sequence like streptavidin e Generally lower nonspecific binding than avidin and streptavidin e Much lower price than streptavidin References 1 Hiller Y et al 1987
9. nm 549Dye 649 Dye Kodak Image Station 535 625 600 700 J J 2000MM Image Station 535 625 600 700 v v 4000MM Bio Rad Molecular Imager 532 635 605 695 J J FX FX Pro Amersham Typhoon 9410 532 633 580 670 J J Typhoon 9400 532 633 580 670 J J Typhoon 9210 532 633 580 670 J J Typhoon 9200 532 633 580 670 J J Storm 830 635 670 Notcompatible V Storm 860 635 670 Notcompatible V Fuji FLA 3000 532 633 570 675 J J FLA 5100 532 633 570 675 J J FLA 8000 532 633 570 675 v v 1 Thermo Scientific DyLight Dye performance has been evaluated with this instrument Compatibility of other instruments is based on manufacturers specifications Figure 10 The Thermo Scientific DyLight 549 649 Western Blotting Kit pro vides lower background and higher signal in two color Western blot detection compared to a competing fluorescent Western blotting kit Proteins were separated in 4 20 Precise Protein Gels and transferred to low fluorescence PVDF membrane The mem branes were blocked overnight in 1 BSA and target proteins were detected following man ufacturer recommended protocols Membranes were imaged with the 12 ng 12 ng MW Marker 6 ng 3ng 1ng 0 5 ng 1ng 3ng D ng 25 ng D c LO N Tubulin TNFa ECL Plex Western Blotting Kit 25 ng 12 ng 6 ng 3ng 1ng 0 5 ng 1ng 3ng D ng 12 ng 25 ng MW Marker Tubulin TNFa Thermo Scientific DyLight 549 649 Western Blotting Kit Typhoon 9410 Ordering Inf
10. 89872 Active Rap1 Pull Down and Detection Kit Ki it it it it For more information or to download product instructions visit www thermo com pierce Far Western Blotting Studying protein interactions by far Western blotting Far Western blotting was developed to screen protein expression libraries with P labeled glutathione S transferase GST fusion protein Far Western blotting is now used to identify protein protein interactions In recent years far Western blotting has been used to determine receptor ligand interactions and to screen libraries for interacting proteins With this method of analysis it is possible to study the effect of post translational modifications on protein protein interactions examine interaction sequences using synthetic peptides as probes and identify protein protein interactions without using antigen specific antibodies For more information on Far Western blotting please refer to the Thermo Scientific Pierce Protein Interaction Handbook 1601666 Protein Interaction Technical Handbook ts The study of protein interactions is vital for understanding protein function within the cell This hand book provides background helpful hints and troubleshooting for methods used to study these interactions including IP and co IP pull downs far Western blotting and crosslinking To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local br
11. Biochem J 248 167 171 Unson M D et al 1999 J Clin Microbiol 37 2153 2157 Wojciechowski M et al 1999 Clin Chem 45 1690 1693 Glover B P and McHenry C S 2001 Cell 105 925 934 Guo Y et al 2001 J Biol Chem 276 45791 45799 Claypool S M et al 2002 J Biol Chem 27 28038 28050 Ordering Information Product Description Features Pkg Size 22831 NeutrAvidin DyLight 405 Conjugated e Excellent photostability 1 mg 22832 NeutrAvidin DyLight 488 Conjugated e Intense emission provides superior sensitivity and requires less conjugate 1mg 22837 NeutrAvidin DyLight 549 Conjugated e Completely stable from pH 4 9 1 mg 22842 NeutrAvidin DyLight 594 Conjugated 1mg 22844 NeutrAvidin DyLight 633 Conjugated 1 mg 22845 NeutrAvidin DyLight 649 Conjugated 1 mg 22848 NeutrAvidin DyLight 680 Conjugated 1 mg 22853 NeutrAvidin DyLight 800 Conjugated 1 mg 31000 NeutrAvidin Biotin Binding Protein e pl that has been reduced to a neutral state 10 mg 31050 NeutrAvidin Biotin Binding Protein e Deglycosylated so lectin binding is reduced to undetectable levels 100 mg e Can be used as a biotin blocking agent in tissues for histochemistry e 11 17 ug biotin bound mg NeutrAvidin Protein 31001 NeutrAvidin Horseradish e Better signal to noise ratio in assay systems 2 mg Peroxidase Conjugated e 1 2 moles HRP mole NeutrAvidin Protein e 3 8 ug biotin bound mg conjugate 31002 NeutrAvidin Alkaline e Lower nonspecific binding
12. e You want to obtain a stronger signal under the conditions you typically use to detect your target protein For more information or to download product instructions visit www thermo com pierce Enzyme Substrates Thermo Scientific Pierce Western Blot Signal Enhancer It s like having an intensifying screen in a bottle There are many ways to increase the sensitivity of a Western blot Some methods are as simple as switching substrates or blocking buffers while others are more time consuming such as optimizing antibody titer or checking for proper protein transfer Those solu tions are detailed in the troubleshooting section of this handbook One of the more certain and easiest ways to increase the sensitivity of any Western blot is to use Pierce Western Blot Signal Enhancer Pierce Western Blot Signal Enhancer does for enzyme substrate based blotting what intensifying screens do for radioactive blotting it increases the signal up to 10 fold or one order of magnitude in only 15 minutes Figures 5 6 The Pierce Western Blot Signal Enhancer membrane treatment Is a simple 15 minute procedure Figure 7 that can be added to your current Western blotting protocol The result is an increase in the intensity of target protein bands on the Western blot or detection of target proteins at a level that could not previously be detected Some protein targets have resulted in a 10 fold increase in band intensity after treatment
13. s 24 hour light emission 10 times longer than other enhanced chemiluminescent substrates for HRP make multiple exposures for publication quality blots e Great sensitivity see bands you ve never been able to see before with femtogram level sensitivity e Maximize your antibody antibodies can be diluted much further when using SuperSignal West Dura Extended Duration Substrate than with other chemiluminescent substrates perform 25 to 50 times more blots e Intense signal generated immediately and easily detected on film or chemiluminescent imager systems e Stable working solution stable for at least 24 hours kit stable for at least one year and shipped at ambient temperature A Thermo Scientific SuperSignal West Dura Substrate 50 25 12 5 6 3 3 1 1 6 0 80 4 0 2 0 1 05 03 013 006 003 ng 5 Minutes with Film B Thermo Scientific SuperSignal West Dura Substrate 50 25 12 5 6 3 3 1 1 6 0 80 4 0 2 0 1 05 03 013 006 003 ng 30 Minutes with Chemilmager 4000 CCD Camera C GE Healthcare ECL Plus Substrate nN N S HPP K FX GE AP 5 Minutes with Film D GE Healthcare ECL Plus Substrate 50 25 12 5 63 3 1 1 6 08 0 4 0 20 1 05 03 013 006 003 ng 15 Minutes with Chemilmager 4000 CCD Camera Figure 5 Better sensitivity and less background The membranes were blocked and incubated with Anti IL 2 antibody 1 g ml After washing the membranes were incubated with secondary antibody 10 ng ml The membra
14. 13 4 per mini gel in USD Transfer efficiency of the Thermo Scientific Pierce Fast Transfer System is comparable to existing methods A549 whole cell lysates were prepared for SDS PAGE and loaded onto a NuPAGE 4 12 Bis Tris Gel 1 0 mm x 10 well using the following protein amounts 16 ug 8 ug 4 ug and 2 ug After elec trophoresis gels were transferred to nitrocellulose membrane using either Pierce Fast Semi Dry Transfer System or the iBlot Dry Blotting System Resulting membranes were probed for PRKDC EGFR PLK1 CDC2 and Cyclophilin B using the Pierce Fast Western Blotting Kit Pierce Fast Semi Dry Transfer Buffer 10X Our methanol free transfer buffer is specially formulated to function with the Thermo Scientific Pierce Semi Dry Transfer Unit Simply dilute the 10X concentrate in water for a freshly prepared transfer buffer for quick and efficient transfer of proteins from gel to membrane of choice Pierce Fast Semi Dry Blotter The new Pierce Fast Semi Dry Blotter provides the means for the efficient 10 minute transfer of proteins from gel to membrane Ordering Information Product Description Pkg Size 35035 Pierce Fast Semi Dry Transfer Buffer 10X 500 ml Sufficient for 50 mini gel transfers 88217 Pierce Fast Semi Dry Blotter lea Thermo Scientific Transfer Buffers BupH Tris Glycine and Tris Buffered Saline Great for Western blots BupH Tris Glycine Buffer Packs Each pack yields 500 ml of 25 mM Tris and
15. 250 pg Lane 2 500 pg Lane 3 1 000 pg and Lane 4 2 000 pg A Untreated Blot 1 2 3 4 5 6 7 ra SS ae ERF ea H A A Figure 6 Enhanced chromogenic detection of identical serial dilutions of IL 6 Panel A before and Panel B after treatment with Thermo Scientific Pierce Western Blot Signal Enhancer Lane 1 100 pg Lane 2 200 pg Lane 3 300 pg Lane 4 400 pg Lane 5 500 pg Lane 6 1 000 pg and Lane 7 5 000 pg ay 5 Rinse membrane with ultrapure water repeat 5 times Ultrapure 1 Ultrapure i b DN 1 Rinse membrane after 2 Incubate membrane with 3 Rinse membrane with transfer with ultrapure water Reagent 1 for 2 minutes ultrapure water on a shaker repeat 5 times 2 Ultrapure y Aa Start your j N detection T protocol 4 Incubate membrane with Total time 15 minutes Reagent 2 for 10 minutes on a shaker Figure 7 Thermo Scientific Pierce Western Blot Signal Enhancer Protocol performed after transfer and before blocking To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor STEP SDS PAGE In a Western blot it is important to block the unreacted sites on the membrane to reduce the amount of nonspecific binding of proteins during subsequent steps in the assay A variety of block ing buffers ranging from milk or normal serum to highly purified proteins have been used to block unreacted sites on a
16. 31992 Goat 31182 31807 31444 81328 Goat S 8 31330 Goat 31232 C8630 Goat 31288 Gott 31287 81634 Horse 30981 3806 l Rabbit 31188 __ 31450 8132931561 Rabbit 31190 8813384 Rabbit 31192 IRI 31331 31559 Rabbit 31194 31813 31455 31332 31555 Rabbit 31196 31456 31333 31557 Rabbit 31198 8 81335 Goat Ffab 30185 __ 38e O 355 GoatFab 30078 GoatFlab 386 GoatFlab Donkey 31998 31821 31458 31345 31568 Goat 31210 31820 31460 31340 31635 Got e S Goat 31212 31822 31462 31342 31583 Goat 31234 31823 31461 31343 315733 Goat 31216 _ _ 33 IBM Mouse 31213 31824 31464 81584 GoatFlab o 81579 GoatF ab 31239 3109 Goat 31220 31830 31470 31350 31629 Goat S 8H Goat 31226 8 862 Goat 3228 80832 8B Rabbit 31218 30834 31219 Rabbit 31240 31840 31480 31360 31627 21125 S 21127 21323 21224 31001 31002 31006 For pricing in the U S visit www thermo com pierce Outside the U S please contact your local branch or distributor Product Rhodamine TexasRed DyLight 405 DyLight 488 DyLight 549 DyLight594 DyLight633 DyLight649 DyLight680 DyLight 750 DyLight 800 C HT T aaa eae po es eee 31660 31498 35o 85507 85510 THE THE es 31685 3100 a ee eee ees eee 31670 3150600 35552 35557 3556000 85565 35568 BT 8555O 35553 35561 35563 35566 35569 Pl T IT T T T T I T I 21724 21726 21
17. 82328 HIF 1A 82366 RHOA 82400 CAVI 82329 HRAS 82367 RIPK1 82401 CCNB1 82330 HSPA1A 82368 SMAD2 82402 CCND1 82331 HSPB1 82369 SMAD2 amp 3 82403 CCNE1 82332 IKBKB 82370 SMAD4 82404 CDC2 82333 ILK 82371 SOS1 82405 CDC25C 82334 IRS1 82372 SP1 82406 CDH1 82335 ITGB1 82373 SRC 82407 CDK2 82336 JUN 82374 STAT1 82408 CDK4 82337 KIF11 82375 STAT3 82409 CDK5 82338 LYN 82376 STAT6 82410 CDK6 82339 MAP2K1 82377 TP53 82411 CDK9 82340 MAP2K3 82378 TTK 82412 CDKN1A 82341 MAP3K7 82379 VIL2 82413 CDKN1IB 82342 MAPK1 amp 3 82380 CDKN2A 82343 MAPK8 82381 p14 Loading Control Antibodies Product Description See Below Each package contains sufficient antibody for 10 mini blots Target Product Target Product Target Product GAPD 82350 Cyclophilin B 82351 Actin 82353 t See patent information on inside back cover For pricing in the U S visit www thermo com pierce Outside the U S please contact your local branch or distributor To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor Electro Transfer Thermo Scientific Pierce Secondary Antibodies are supplied lyophilized or in solution and are stable for at least 1 year when stored as directed Lyophilized antibodies are formatted to provide a buffered and stabilized solution when reconstituted in water at approximately 1 mg ml individual product inserts specify lot specific values Antibodies provided in solu
18. Animal Protein Non Animal Protein Blocking Buffer Blocker X Blocker Y 1 Minute B vroad ET PVDF Nitrocellulose PVDF Nitrocellulose PVDF Film Exposure 1 Minute Film Exposure Figure 2 Thermo Scientific Protein Free Blocking Buffer efficiently blocks Western blotting membranes Jurkat apoptotic lysate Lane 1 0 25 ug Lane 2 0 50 ug was separated in 4 20 Tris glycine gels and transferred to nitrocel lulose or PVDF membranes The membranes were blocked for 1 hour at RT with the indicated blocking buffer probed with mouse anti PARP 0 25 ug ml followed by goat anti mouse HRP 4 ng ml and detected by SuperSignal West Dura Chemiluminescent Substrate For more information or to download product instructions visit www thermo com pierce Enzyme Substrates Thermo Scientific StartingBlock Blocking Buffer Simplify the selection of a blocker for Western blot and ELISA applications Although no blocking buffer is ideal for every system you can improve the odds dramatically with StartingBlock Blocking Buffer because It is compatible with the widest variety of antibodies For example StartingBlock Blocking Buffers are compatible with biotin containing systems while milk based protein blockers interfere StartingBlock Buffers rarely cross react with rabbit antibodies while many other blockers do StartingBlock Blocking Buffers are also free of potentially interfering serum proteins StartingBlock Blocking Buffers offe
19. BLOCK Blocking Buffer No mammalian proteins reducing the risk of nonspecific interaction Highlights e Made from steelhead salmon serum e Functions as a universal blocker e Offers reduced background e Can be diluted up to 1 10 with buffer References Hypolite J A et al 2001 Am J Physiol Cell Physiol 280 C254 264 Wang L et al 2002 J Clin Invest 110 1175 1184 Ordering Information Product Description Pkg Size 37527 SEA BLOCK Blocking Buffer 500 ml Blocker Casein Ready to use solution 1 w v of Hammersten Grade casein for blocking nonspecific sites Highlights e Preformulated for ease of use e Use when skim milk produces high background e Thimerosal free formulation STEP Blocki Ordering Information Product Description Pkg Size 37532 Blocker Casein in TBS 1L 1 w v Casein Hammersten Grade in TBS Contains Kathon Antimicrobial Reagent as preservative pH 7 4 37528 Blocker Casein in PBS 1L 1 w v Casein Hammersten Grade in PBS Contains Kathon Antimicrobial Reagent as preservative pH 7 4 Blocker BLOTTO Ready to use blocking buffer made of nonfat dry milk Highlights e Preformulated for ease of use e Available in TBS Buffer Ordering Information Product Description Pkg Size 37530 Blocker BLOTTO in TBS LE 5 w v nonfat powdered milk in TBS 0 01 Anti foam A contains Kathon Antimicrobial Reagent as preservative pH 7 4 e Anti foaming agent added e
20. Femto Chemiluminescent Substrate Product 34095 detection limits as low as 1 femtogram are possible because the enhancers in this substrate greatly intensify the emitted light and extend the signal duration Chemiluminescent substrates differ from other substrates in that the light detected is a transient product of the reaction that is only present while the enzyme substrate reaction is occurring This is in contrast to substrates that produce a stable colored product these colored precipitates remain on the membrane after the enzyme substrate reaction has terminated On a chemiluminescent Western blot the substrate is the limiting reagent in the reaction as it is exhausted light production decreases and eventually ceases A well optimized procedure using the proper antibody dilutions will produce a stable output of light for several hours allowing consis tent and sensitive detection of proteins When the antibody is not diluted sufficiently a stable output of light will never be achieved Too much enzyme in the system will rapidly oxidize the substrate and terminate the signal This is the single greatest cause of symp toms such as variability dark background with clear bands and decreased sensitivity in Western blotting experiments with chemi luminescence To avoid this problem it is crucial to optimize the amount of antibody used for detection Antibody suppliers typically suggest a dilution range for using their antibody on a We
21. Merthiolate free formulation Blocker BSA For all blocking applications Highlights e 10 solutions of high quality bovine serum albumin e Concentrated formulation saves storage space e No powder to dissolve ready to dilute liquid concentrate Ordering Information Product Description Pkg Size 37525 Blocker BSA in PBS 10X 200 ml 37520 Blocker BSA in TBS 10X 125 ml Surfact Amps 20 Purified Detergent Solution Specially purified form of Tween 20 Detergent Highlights e Guaranteed lt 1 milliequivalent of peroxides and carbonyl in a 10 solution e Enhances signal to background ratio Ordering Information Pkg Size 6 x 10 ml Product Description 28320 Surfact Amps 20 Purified Detergent Solution For more information or to download product instructions visit www thermo com pierce Enzyme Substrates Like other immunoassay procedures Western blotting consists of a series of incubations with different immunochemical reagents separated by wash steps Washing steps are necessary to remove unbound reagents and reduce background thereby increasing the signal to noise ratio Insufficient washing produces high back ground while excessive washing may result in decreased sensitivity caused by elution of the antibody and or antigen from the blot As with other steps in performing a Western blot a variety of buffers may be used Occasionally washing is performed in a physiological buffer such as Tris buffere
22. SDS PAGE Each protein in the mixture fluoresces at two wavelengths in the near infrared region of the spectrum to enable one or two color detection with the LI COR Odyssey Infrared Markers only or common CCD instruments The markers are compatible with Western blotting and can be detected by virtually any in gel staining method The DyLight Fluorescent Protein Molecular Weight Markers consist of nine proteins with MW in the range of 6K to 200K Figure 2 To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor STEP STEP SDS PAG Electro Ira r Highlights e Excitation emission maxima 557 570 and 652 673 or 682 715 and 710 194 e Easily multiplexed two excitation and emission maxima enable one or two color fluorescent detection e Saves time no awkward marking or overlay procedures e Fluorescent and colorimetric detect in gel or on membrane e Instrument compatible spectra are compatible with LI COR Odyssey infrared markers only and CCD instruments e Photostable capture multiple images with no decrease in fluorescent intensity eee R 9 Myosin 200K Phosphorylase B 97K BSA 66K Protein A 45K Protein L 36K Peanut Agglutinin 27K Trypsin Inhibitor 20K Lysozyme 14K Aprotinin 6K T e Ld m Figure 2 Detection methods for the DyLight Fluorescent and Infrared Markers Pane
23. Scientific Pierce Prestained Marker Protein molecular weights Each tube of the Pierce Marker consists of a stabilized and lyophi lyzed formulation of seven proteins ranging from 16 5K to 210K Each protein in the mixture is proportioned to yield uniform band intensities Two specially modified bands one red one violet serve as references for the order of the marker proteins These are representative molecular weight values The covalently bound dye and enzyme alter the apparent molecular weight MW of the component proteins relative to their unstained counterparts Lot specific MW values are provided with each package Ordering Information Product Description Pkg Size 26681 Pierce Blue Prestained Protein 1 x 48 Molecular Weight Marker Mix microtube Sufficient material for loading 48 96 gel lanes plate 26685 Pierce Blue Prestained Protein 5 x 48 Molecular Weight Marker Mix microtube Sufficient material for loading 240 480 gel lanes plates 26691 Pierce 3 Color Prestained Protein 1 x 48 Molecular Weight Marker Mix microtube Sufficient material for loading 48 96 plate gel lanes in a 6 x 8 microtube plate format Thermo Scientific DyLight Fluorescent and Infrared MW Markers One or two color fluorescent detection with one protein MW marker DyLight Fluorescent and Infrared Protein Molecular Weight Markers are optimized for direct visualization of marker proteins after sodium dodecyl sulfate polyacrylamide gel electrophoresis
24. Sensitizer 250 ml PVDF membrane pre treatment agent Pierce Reversible Stain 250 ml A broad spectrum stain for proteins transferred to PVDF membrane Pierce Destain 1 000 ml Enhances protein band detection by eliminating background stain Pierce Stain Eraser 500 ml Reverses protein band staining on demand Reagent grade methanol required but not supplied supplements the Destain and Stain Eraser formulations Table 1 Comparison of Thermo Scientific Pierce Reversible Protein Stain with Ponceau S Stain Thermo Scientific Pierce Reversible Protein Stain Ponceau S Reversible Stain e Weak binding low sensitivity general protein stain e Tight binding higher sensitivity general protein stain e Detection limit 250 ng e Detection limit 25 50 ng e Red bands are difficult to photograph e Turquoise blue bands are photographed easily e Stained protein bands fade within hours e Turquoise bands do not fade over time but they can be reversed e Typical staining time 5 minutes e Typical staining time 60 seconds e Background eliminated quickly with low pH wash To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor STEP STEP STEP STEP Formul SDS PAGE Electro Ira r Blocki Wash Buff Thermo Scientific Pierce Antibody Extender NC Highlights T e Achieves equivalent signal while using less antibody uses Get t
25. blocked for 1 hour at RT with shaking in Blocker Casein in TBS 1 BSA in TBS 37530 Blocker Vv v U Y v SuperBlock Blocking Buffer in TBS or 5 nonfat milk in TBS Tween 20 0 05 BLOTTO in TBS was added to all blocking buffers The membranes were then incubated 37570 Protein Free Vn Yv with the appropriate primary antibody at 0 5 ug ml prepared in the different TBS blocking solutions for one hour at RT with shaking Each membrane strip was washed with TBS followed by a one hour incubation in HRP conjugated goat anti mouse antibody prepared in the different blocking buffers at 25 ng ml The membranes were washed with TBS A working solution of SuperSignal West Pico Chemiluminescent Substrate was prepared and added to each membrane Blocking Buffer 37571 Protein Free v Y Y Y T20 TBS Blocking Buffer for 5 minutes The membranes were placed in sheet protectors and exposed to 37572 Protein Free vY Y v film for 30 seconds and 5 minutes as indicated The film was developed per the PBS manufacturer s instructions Blocking Buffer 37573 Protein Free T20 v Vv Vv PBS Blocking Buffer To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor STEP STEP STEP STEP Formul SDS PAGE Electro Transfer Wash Buff Thermo Scientific Protein Free Blocking Buffers Ordering Information Eliminate or minimize cross reactivity to reduce background and Product Descri
26. byproduct HRP functions optimally at a near neutral pH and can be inhibited by cyanides sulfides and azides Antibody HRP conjugates are superior to antibody AP con jugates with respect to the specific activities of both the enzyme and antibody In addition its high turnover rate good stability low cost and wide availability of substrates make HRP the enzyme of choice for most applications Table 3 Key to abbreviations for individual species Bv Bovine Gu Guinea Pig Hs Horse Rt Rat Ch Chicken Ha Hamster Ms Mouse Sh Sheep Gt Goat Hn Human Rb Rabbit Sw Swine Affinity purified antibodies are available unconjugated or conjugated with biotin alkaline phosphatase horseradish peroxidase fluorescein rhodamine and DyLight Dyes F ab fragments of antibodies to immunoglobulins are also available in unconjugated or conjugated forms These F ab fragments of antibodies are especially useful in assays in which binding between the Fc portions of antibodies and Fc receptor bearing cells must be eliminated Polyclonal antibodies are purified by immunoaffinity chromatography to eliminate nonspecific antibodies resulting in high sensitivity and specificity and low background The purification process involves an elution procedure yielding antibodies with high avidity These antibodies exhibit maximal binding to antigens and minimal cross reactivity to other molecules Conjugated antibodies are affinity purified b
27. efficiently transferred to a membrane Thermo Scientific Pierce In Gel Detection Technology allows positive identification of proteins directly in a gel Product s 33500 33505 33510 and 33515 Blocking Block nonspecific sites e Protein free Blocking Buffer Product s 37570 37571 37572 and 37573 e StartingBlock Blocking Buffer in PBS Product 37538 and in TBS Product 37542 e StartingBlock T20 Blocking Buffer Contains 0 05 Tween 20 in PBS Product 37539 or TBS Product 37543 e SuperBlock Buffer in PBS Product 37515 and 37518 and in TBS Product 37535 e SuperBlock T20 Blocking Buffer Contains 0 05 Tween 20 in PBS Product 37516 or TBS Product 37536 e SuperBlock Blocking Buffer Blotting in PBS Product 37517 and in TBS Product 37537 e Casein in PBS Product 37528 and in TBS Product 37532 e BSA in PBS Product 37525 and in TBS Product 37520 e SEA BLOCK Buffer Product 37527 e BLOTTO in TBS Product 37530 w STEP 4A Formulate Wash Buffers Choose a buffer e Phosphate Buffered Saline PBS Product s 28372 and 28348 e Tris Buffered Saline TBS Product s 28376 28379 and 28358 e Modified Dulbecco s PBS Product s 28374 and 28344 e Carbonate Bicarbonate Buffer Packs Product 28382 e MES Buffered Saline Product 28390 e BupH Borate Buffer Packs Product s 28384 and 28341 e BupH Citrate Carbo
28. ml Thermo Scientific Pierce Substrate Pierce ECL Substrate SuperSignal West Pico Substrate 1 20 000 1 100 000 or 10 50 ng ml SuperSignal 1 100 000 1 500 000 or 2 0 10 ng ml West Femto Substrate SuperSignal 1 50 000 1 250 000 or 4 0 20 ng ml West Dura Substrate Lumi Phos WB Substrate 1 5 000 1 25 000 or 40 200 ng ml For more information or to download product instructions visit www thermo com pierce Enzyme Substrates funooysajqnosy 8 Wash the membrane again as described in Step 6 9 Prepare the substrate working solution by mixing equal volumes of the Luminol Enhancer Solution and the Stable Peroxide Solution Prepare a sufficient volume to ensure that the blot is completely wetted with substrate and the blot does not dry out during incubation Recommended volume 0 1 ml cm of blot surface 10 Incubate the membrane in the SuperSignal West Pico Substrate Working Solution for 5 minutes 11 Remove the membrane from the substrate and place in a plastic sheet protector or other protective wrap 12 Place the blot against the film protein side up and expose Any standard or enhanced autoradiographic film can be used A recommended first exposure is 30 60 seconds Exposure time can be varied to obtain optimum results Alternatively use a CCD camera or other imaging device however these devices may require longer exposure times 13 On an optimized blot the SuperSignal West Pico Subs
29. molecular weight of 214 1 and yields a brown precipitate in the presence of HRP and peroxide The brown insoluble product can be readily chelated with osmium tetroxide This property makes DAB ideal for electron microscopy The color produced by DAB can be intensified with the addition of metals such as nickel copper silver and cobalt that form complexes The color produced by the metal complexes Is darker than the color produced by DAB alone enhancing the sensitivity in staining applications Ordering Information Product Description Pkg Size 34002 Pierce DAB Substrate Kit 275 ml Includes DAB 10X 25 ml Stable Peroxide Buffer 250 ml 34065 Pierce Metal Enhanced DAB 275 ml Substrate Kit Includes 10X Metal Enhanced DAB 25 ml Stable Peroxide Buffer 250 ml The individual benefits of 4 CN and DAB are often combined into a single substrate mixture CN DAB Substrate The CN DAB Substrate has excellent sensitivity yielding a dark black precipi tate that photographs well The CN DAB Substrate works well in Western blotting and dot blotting applications Ordering Information Product Description Pkg Size 34000 Pierce CN DAB Substrate Kit 275 ml Includes CN DAB 10X 25 ml Stable Peroxide Buffer 250 ml Substrates for Alkaline Phosphatase NBT with a molecular weight of 817 6 is a member of a class of heterocyclic organic compounds known as tetrazolium salts Upon reduction the compound yields NBT formazan a highly colored
30. of recombinant bovine TNF Product RBOTNFAI were prepared and electrophoresed The proteins were transferred to nitrocel lulose membranes Product 88025 Membranes were blocked with 5 skim milk and then incubated with rabbit anti bovine TNF o at 4 ug ml The membranes were washed and then incubated with 0 4 ug ml of HRP conjugated Goat Anti Rabbit IgG Product 31460 and then washed again Working solutions of the substrates were prepared according to the manufacturers instruc tions and added to the membranes for 1 minute The membranes were placed in plastic sheet protectors and exposed to Hyperfilm Film GE Healthcare Piscataway NJ Highlights e Half the price of other ECL Substrates low overhead and a commitment to customer value enables us to offer this product for half the price other companies charge these claims are based on the 2007 U S list prices e No optimization required switch to our ECL substrate without the need for optimization or protocol changes s A product you can rely on we put both our strong technical support and reputation behind this product Ordering Information Product Description Pkg Size 32106 Pierce ECL Western Blotting Substrate 500 ml kit 32209 Pierce ECL Western Blotting Substrate 250 ml kit 32109 Pierce ECL Western Blotting Substrate 50 ml kit To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor
31. office or distributor STEP STEP SDS PAGE Electro Tra r When energy in the form of light is released from a substance because of a chemical reaction the process is called chemiluminescence Luminol is one of the most widely used chemiluminescent reagents and its oxidation by peroxide results in creation of an excited state product called 3 aminophthalate This product decays to a lower energy state by releasing photons of light Figure 2 0 0 gt 0 CE OF Or Me 425 nm NH 0 NH 0 NH 0 Figure 2 Luminol is oxidized in the presence of HRP and hydrogen peroxide to form an excited state product 3 aminophthalate The 3 aminophthalate emits light at 425 nm as it decays to the ground state Chemiluminescent substrates have steadily gained in popularity because they offer several advantages over other detection methods Table 1 These advantages have allowed chemilumi nescence to become the detection method of choice in most protein laboratories Using chemiluminescence allows multiple exposures to obtain the best image The detection reagents can be removed and the entire blot reprobed to visualize another protein or to optimize detection of the first protein A large linear response range allows detection and quantitation for a large range of protein concentrations Most importantly chemiluminescence yields the greatest sensitivity of any available detection method Using HRP as the enzyme label and SuperSignal West
32. preparation tracking or refrigeration hassles 3 Move forward with your research by eliminating re tests from buffer problems BupH Phosphate Buffered Saline Packs PBS Great wash buffer for Western blots Each pack yields 500 ml of 0 1 M phosphate 0 15 M sodium chloride pH 7 0 when dissolved in 500 ml deionized water 20 L total Ordering Information Product Description Pkg Size 28372 BupH Phosphate Buffered 40 pack Saline Packs 28348 20X Phosphate Buffered Saline 500 ml 28352 20X PBS Tween 20 500 ml BupH Tris Buffered Saline TBS Great wash buffer for Western blots Each pack yields 500 ml of 25 mM Tris 0 15 M sodium chloride pH 7 2 when dissolved in 500 ml deionized water 10 pack makes 5 L total 40 pack makes 20 L total Ordering Information Product Description Pkg Size 28380 BupH Tris Glycine Buffer Packs 40 pack 28376 BupH Tris Buffered Saline Packs 40 pack 28379 BupH Tris Buffered Saline Packs 10 pack Surfact Amps 20 Purified Detergent Solution Specially purified form of Tween 20 Detergent Highlights e Can be added to PBS or TBS wash buffers to improve performance e Guaranteed lt 1 milliequivalent of peroxides and carbonyl in a 10 solution e Enhances signal to background ratio Ordering Information Pkg Size 6 x 10 ml Product Description 28320 Surfact Amps 20 To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor
33. raised the host species For example if the primary antibody is a mouse monoclonal antibody the secondary antibody must be an anti mouse antibody obtained from a host other than the mouse The host species of the secondary antibody often will not affect the experiment However secondary antibodies are available from many host species and if a secondary antibody causes high background in a particular assay another host spe cies may be chosen Another option to reduce background is to use a secondary antibody that has been pre adsorbed to serum proteins from other species This pre adsorption process removes antibodies that have the potential to cross react with serum pro teins including antibodies from those species To expedite the process of choosing the appropriate secondary antibody visit the Secondary Antibody Selection Guide on our website Antibody solutions for Western blotting are typically diluted from 1 100 to 1 500 000 beginning from a 1 mg ml stock solution The optimal dilution of a given antibody with a particular detection system must be determined experimentally More sensitive detec tion systems require less antibody which can result in substantial savings on antibody costs and allow a limited supply of antibody to be used for many experiments It also produces a side benefit of reduced background because the limited amount of antibody is specific for the target with the highest affinity Antibody dilutions are typicall
34. tissue staining Ordering Information Product Description 21122 21125 21135 21126 21124 21127 21324 21323 21224 21724 21624 21627 21629 21120 21831 21832 21837 21842 21844 21845 21848 21850 21851 Streptavidin Streptavidin Streptavidin Horseradish Peroxidase Conjugated Horseradish Peroxidase Conjugated Horseradish Peroxidase Conjugated Alkaline Phosphatase Conjugated Alkaline Phosphatase Conjugated Fluorescein FITC Conjugated Rhodamine TRITC Conjugated Texas Red Conjugated R Phycoerythrin Conjugated Allophycocyanin Conjugated e Ex Em 650 nm and 660 nm Hydrazide Activated Streptavidin DyLight 405 Conjugated Ex Em 400 420 lt Excellent photostability Features e Lyophilized stable powder e No carbohydrate e Much less soluble in water than avidin e 13 22 ug biotin bound mg of protein e Recombinant e 1 2 moles HRP mole streptavidin e gt 100 peroxidase units mg conjugate e Lyophilized stable powder e 6 9 ug biotin bound mg conjugate e gt 3 ug biotin bound mg conjugate e gt 100 phosphatase units mg conjugate e Fluorescently labeled streptavidin e Ex Em 490 nm and 520 nm e 3 5 moles FITC mole streptavidin e Fluorescently labeled streptavidin e Excitation 515 520 nm and 550 555 nm e Emission 575 nm e 1 3 moles TRITC mole streptavidin e Fluorescently labeled streptavidin e Ex Em 595 nm and 615 nm e Fluorescently labeled strep
35. visualizing Substrates such as TMB 3 37 5 5 tetramethylbenzi dine 4 CN 4 chloro 1 naphthol and DAB 3 3 diaminobenzidine tetrahydrochloride are available for use with HRP For use with AP NBT nitro blue tetrazolium chloride BCIP 5 bromo 4 chloro 3 indolylphosphate p toluidine salt and Fast Red naphthol AS MX phosphate Fast Red TR Salt are available The performance of a particular substrate may vary dramatically when obtained from different suppliers because performance can be affected by the concentration and purity of the substrate and by other additives and buffer components that are a part of the formulation STEP STEP Formul Wash Buff Peroxide must be added to a substrate for colorimetric detection with HRP Because of its extremely short shelf life at the desired concentration hydrogen peroxide traditionally was added to a buffer along with the substrate immediately before use As a result these substrates typically have a useful shelf life of only a few hours Many of our precipitating HRP substrates are sup plied with or come prepared in Stable Peroxide Substrate Buffer Product 34062 The Stable Peroxide Substrate Buffer is a 10X concentrate that offers several advantages It is less corrosive than the traditional 30 stock solution of hydrogen peroxide and because fewer preparation steps are involved it provides more consistent results Although the Stable Peroxide Substrate Buff
36. with the Western Blot Signal Enhancer compared to the typical detection protocol without treatment Highlights Enhances chemiluminescent fluorescent and colorimetric detection up to 10 fold e Treatment with Western Blot Signal Enhancer can boost the band intensity from three to 10 fold regardless of which substrate is used Enhances detection of targets transferred to either nitrocellulose or PVDF independent of membrane pore size e Works with the most commonly used Western blotting membranes e Signal intensity has been increased with targets such as mouse IL 6 p53 NF B BRCA1 and EGF Room temperature stable ready to use reagents e No thawing formulating or diluting necessary 15 minute protocol e Optimized to save time and improve detection capability of your specific analyte Ordering Information Product Description Pkg Size 21050 Pierce Western Blot Signal Enhancer Kit Sufficient reagent for ten 10 cm x 10 cm blots Includes Enhancer Reagent 1 250 ml Enhancer Reagent 2 250 ml Signal enhancement of proteins on PVDF membrane has been shown to be variable from no significant enhancement for some proteins to several fold enhancement for others A Untreated Blot 1 2 3 4 B Treated Blot 1 2 3 4 Figure 5 Enhanced chemiluminescent detection of identical serial dilutions of IL 6 Panel A before and Panel B after treatment with Thermo Scientific Pierce Western Blot Signal Enhancer Lane 1
37. 0 12 30 ul 10 gels 25240 8 15 25 ul 10 gels 25241 10 15 25 ul 10 gels 25242 12 15 25 ul 10 gels 25243 8 16 15 25 ul 10 gels 25244 4 20 15 25 ul 10 gels Tris HEPES SDS Running Buffer Required running buffer for use with Precise Gels Precise Protein Gels use a unique Tris HEPES SDS running buffer to improve band resolution and reduce run time The buffer can be made according to the recipe provided in the Precise Gel product instructions or purchased premixed Ordering Information Product Description Pkg Size 28398 BupH Tris HEPES SDS Running Buffer 10 pack Each pack yields 500 ml of 100 mM Tris 100 mM HEPES 3 mM SDS pH 8 0 25 when dissolved in 500 ml distilled water 5 L total 28368 20X Tris HEPES SDS Buffer 0 5 ml 28362 10X Tris Glycine SDS Buffer 1L Electrophoresis Technical Handbook This 44 page reference guide provides information to improve the speed convenience and sensitivity of your protein gel electrophore sis and staining applications The handbook covers all aspects of electrophoresis from sample and gel preparation to choice of molecular weight markers In addition it contains an extensive section on protein gel staining techniques and products For more information or to download product instructions visit www thermo com pierce Enzyme Substrates Another method of verifying target protein transfer is to check the transfer of the molecular weight MW markers We offe
38. 0 5 ml 0 5 ml 0 5 ml 0 5 ml 0 5 ml 0 5 ml 0 5 ml 0 5 ml 0 5 ml Electro Transfer Using Antibodies A Laboratory Manual Few technical manuals have become standards in bioresearch like Antibodies A Laboratory Manual by Ed Harlow and David Lane which has enjoyed that status for more than a decade Using Antibodies The authors however have raised the standard with the publication of their book Using Antibodies A Laboratory Manual Harlow and Lane have completely revised their guide for using antibody reagents in the laboratory Chapters have been entirely rewritten reorga nized and updated to provide background context and step by step instructions for techniques ranging from choosing the right antibody and handling it correctly to the proper methods for characterizing antigens in cells and solutions They ve also added new chapters on tagging proteins and epitope mapping Rather than presenting an array of solutions for working with antibodies and antigens Using Antibodies identifies the best approach to specific problems These recommendations include more detail in the protocols extensive advice on avoiding and solving problems information regarding proper controls and thorough illustration of theory methods and results The book also includes a bonus a set of portable protocols that include step by step instructions for the most frequently used and es sential techniques The protocols are printed o
39. 0 linked serine or threonine e No cross reactivity with the 0 GIcNAc linkage 1 2 3 4 5 6 78 N Figure 9 Western blot detection of 0 GilcNAc modified proteins after SDS PAGE Lanes 1 4 are proteins from the Jurkat cell extract Lanes 5 6 and 7 are the negative controls ovalbumin 5 ug fetuin 5 ug and O B GalNAc modified BSA 10 ng Lane 8 is 0 B GlcNAc modified BSA 5 ng positive control The and refer to plus and minus treatment with PUGNAc and glucosamine and M represents the molecular weight marker Pierce Blue Prestained Protein Molecular Weight Marker Mix Product 26681 STEP STEP Formul Wash Buff CH 0o C NH H H OH H C H HO uc H i H C 0 CH20H 0 H H H B 0 GIcNAc Modified Serine Threonine in Peptide Linkage Ordering Information Product Description Pkg Size 24565 0 GIcNAc Western Blot Kit Detection Kit Sufficient material to develop up to 10 mini blots Includes M PER Mammalian 25 ml Protein Extraction Reagent Dilution Buffer 10X Blocking Buffer 2 x 50 ml BupH Phosphate Buffered Saline 17 packs Surfact Amps 20 3x 10 ml 10 Tween 20 Solution Anti O GlcNAc Monoclonal Antibody 100 ul MAb CTD 110 6 in ascites Goat anti Mouse IgM u 75 ug HRP Conjugate SuperSignal West Dura Extended 100 ml Duration Substrate Note This Western blot kit is shipped in a single box as a two part kit Part A contains some components that requi
40. 000 Secondary 1 20 000 1 100 000 Secondary 1 50 000 1 250 000 lt Secondary 1 100 000 1 500 000 STEP STEP Formul Wash Buff 100 fg 10 fg 1 fg Figure 6 True femtogram detection of IkBa using Thermo Scientific SuperSignal West Femto Maximum Sensitivity Substrate Serially diluted sam ples from 100 to 1 fg were run on 4 20 Precise Precast Gels The protein was then transferred to PVDF membrane and blocked with StartingBlock Blocking Buffer for 1 hour at room temperature RT The blot was incubated in Rabbit Anti l kBo 1 mg ml at 1 1 000 dilution overnight at 4 C followed by incubation in Goat Anti Rabbit HRP 1 mg ml at 1 200 000 dilution for 1 hour at RT The membrane was exposed to CL XPosure Film for 1 minute Ordering Information Product Description Pkg Size 34096 SuperSignal West Femto 200 ml Maximum Sensitivity Substrate Sufficient materials for 2000 cm membrane Includes Luminol Enhancer Solution 100 ml Stable Peroxide Solution 100 ml 34095 SuperSignal West Femto 100 ml Maximum Sensitivity Substrate Sufficient materials for 1 000 cm membrane Includes Luminol Enhancer Solution 50 ml Stable Peroxide Solution 50 ml 34094 SuperSignal West Femto 20 ml Maximum Sensitivity Substrate Trial Kit Sufficient materials for 200 cm membrane Includes Luminol Enhancer Solution 10 ml Stable Peroxide Solution 10 ml References Adilakshmi T and Laine R O 2002 J Biol Chem 27
41. 1 2 3 4 A Control B Thermo Scientific Pierce Reversible Stain Figure 4 Immunoblot analysis of GST by chemiluminescent detection after Thermo Scientific Pierce Reversible Staining destaining and stain reversal Different amounts of purified GST protein were applied to two 10 Tris glycine SDS polyacrylamide gels and electroblotted to nitrocellulose membranes The control membrane Panel A was not treated Panel B was subjected to the staining detaining and stain erasing protocol of the Pierce Kit Both mem branes were probed with anti GST incubated with goat anti rabbit IgG HRP conjugate and detected using SuperSignal West Dura Substrate Product 34075 Lane 1 125 pg Lane 2 250 pg Lane 3 500 pg and Lane 4 1 ng Ordering Information Product Description Pkg Size 24580 Pierce Reversible Protein Stain Kit for Kit Nitrocellulose Membranes Sufficient material to stain protein and reverse the stain from 10 8 cm x 8 cm nitrocellulose membranes Includes Pierce Reversible Stain 250 ml A broad spectrum stain for proteins transferred to nitrocellulose membranes Pierce Destain 1 000 ml Enhances protein band detection by eliminating background stain Pierce Stain Eraser 500 ml Reverses protein band staining on demand 24585 Pierce Reversible Protein Stain Kit for Kit Polyvinylidene Difluoride Membrane Sufficient material to stain protein and reverse the stain from 10 8 cm x 8 cm PVDF membranes Includes Pierce
42. 192 mM glycine at a pH of approximately 8 when dissolved in 400 ml deionized water and 100 ml of methanol 20 L total STEP BupH Tris Buffered Saline Packs Each pack yields 500 ml of 25 mM Tris 0 15 M NaCl pH 7 2 when dissolved in 500 ml deionized water 10 pack makes 5 L total 40 pack makes 20 L total Pierce Methanol Free Transfer Buffer 10X Our Methanol Free Tank Transfer Buffer does not require cooling Simply dilute the 10X solution with water and use directly Product Description 28380 28376 28379 35040 BupH Tris Glycine Buffer Packs BupH Tris Buffered Saline Packs BupH Tris Buffered Saline Packs Pierce Methanol Free Transfer Buffer 10X Formul Wash Buff STEP Ordering Information Pkg Size 40 pack 40 pack 10 pack 5L Complementary Products Transfer Membranes Nitrocellulose Membranes Product Description 88013 88018 88014 88024 77012 88025 77010 Nitrocellulose Membrane 0 2 pm 7 9 cm x 10 5 cm Nitrocellulose Membrane 0 45 pm 33 cmx3 m Nitrocellulose Membrane 0 45 pm 7 9 cm x 10 5 cm Minimum 87 sheets when cut to 7 9 cm x 10 5 cm minimum 52 sheets when cut to 11 5 cm x 12 5 cm Nitrocellulose Membrane 0 2 pm 8 cm x 8 cm Nitrocellulose Membrane 0 2 pm 8cmx12cm Nitrocellulose Membrane 0 45 pm 8 cm x 8 cm Nitrocellulose Membrane 0 45 pm 8 cm x 12 cm Polyvinylidene Difluoride PVDF Membranes Product Description 22860
43. 7 4147 4151 Conti L R et al 2001 J Biol Chem 276 41270 41278 Guo Y et al 2001 J Biol Chem 276 45791 45799 SuperSignal West Dura Extended Duration Substrate SuperSignal West Femto Maximum Sensitivity Substrate e The most sensitive chemiluminescent substrate for HRP detection available e Extended signal duration is ideal for use with imaging equipment e Mid femtogram 10 e High zeptomole 10 e Low femtogram 10 e Mid zeptomole 10 e 24 hours e 8 hours e Primary 1 1 000 1 50 000 e Primary 1 5 000 1 100 000 e 8 hours e 24 hours e 1 year at RT e 1 year at 4 C or 6 months at RT Lower detection limits were determined using Streptavidin HRP or Biotinylated HRP as the ligand Please follow recommended antibody dilutions SuperSignal Substrates are much more sensitive than other substrates so it is critical that you follow these guidelines Failure to do so could result in unsatisfactory results Dilutions are from a 1 mg ml stock solution For more information or to download product instructions visit www thermo com pierce Lumi Phos WB Chemiluminescent Substrate A chemiluminescent substrate for AP detection that provides the best of both worlds high sensitivity and low background Lumi Phos WB Substrate provides sensitivity in the low picogram range Figure 7 enabling you to detect mere attomoles of your target ligand Lumi Phos WB Substrate also produces less b
44. 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor N mmi e rr U SDS PAGE Electro Trangrer SuperBlock Dry Blend TBS Blocking Buffer Delivers the ultimate in space saving convenience Highlights e Delivers even more economy and stability e Each pouch reconstitutes to form 200 ml of SuperBlock Blocking Buffer in TBS e Room temperature storage small packaging takes up minimal shelf space References Ikeda K et al 2003 J Biol Chem 278 7725 7734 Leclerc G J and Barredo J C 2001 Clin Cancer Res 7 942 951 Subbarayan V et al 2001 Cancer Res 61 2720 276 Walters R W et al 2002 Cell 100 789 799 Ordering Information Product Description Pkg Size 37515 SuperBlock PBS Blocking Buffer 1L 37516 SuperBlock T20 PBS Blocking Buffer 1L Contains 0 05 Tween 20 Detergent 37518 SuperBlock PBS Blocking Buffer 5L 37535 SuperBlock TBS Blocking Buffer 1L 37536 SuperBlock T20 TBS Blocking Buffer 1L Contains 0 05 Tween 20 Detergent 37517 SuperBlock PBS Blocking Buffer Blotting 1 L 37537 SuperBlock TBS Blocking Buffer Blotting 1 L 37545 SuperBlock TBS Blocking Buffer 5 pouches Dry Blend Blocking Buffer Each pouch yields 200 ml when reconstituted Formulated for precipitating enzyme substrates Added ingredient to keep precipitate from flaking Not recommended for chemiluminescent substrates SEA
45. 83 Anal Biochem 171 1 32 Gitlin G et al 1987 Biochem J 242 923 926 Bruch R C and White III H B 1982 Biochemistry 21 5334 5341 Zuk PA and Elferink L A 2000 J Biol Chem 275 26754 26764 Avidin is more soluble than streptavidin and has an Isoelectric point pl of 10 5 It is also more economical than streptavidin and is commonly used in signal amplification systems such as the ABC system Ordering Information Product Description 21121 21128 21123 29994 21321 21221 21021 Avidin Avidin Horseradish Peroxidase Conjugated Horseradish Peroxidase Conjugated Alkaline Phosphatase Conjugated Fluorescein FITC Conjugated R Phycoerythrin Conjugated Features e Hen egg white glycoprotein affinity purified salt free lyophilized powder e 11 14 ug biotin bound mg avidin e Isoelectric point of 10 10 5 e Stable over a wide range of pH and temperatures e Purified using special affinity techniques to eliminate nucleic acids e 1 2 moles HRP mole avidin e 5 10 ug biotin bound mg protein e gt 80 peroxidase units mg protein e Homogeneous by SDS PAGE e Purified using special affinity techniques to eliminate nucleic acids e 1 mole alkaline phosphatase mole avidin e One unit 1 0 micromole of p nitrophenol liberated from p nitrophenylphosphate per minute at 37 C pH 9 5 e Fluorescent labeled avidin e Ex Em 490 nm and 520 nm e No free fluorescein e 3 5 m
46. 881 21832 21837 21842 2184 21845 21848 21850 21851 Oooo O o 22881 22882 22837 22842 2284 22845 22848 22853 To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor 26 Enzyme Substrates Fluorophore Conjugated Secondary Antibodies Traditional FITC fluorescein and other conjugates for cell Characteristics of traditional fluors sorting and other methods Fluorophore Emission Color Ex Em nm et Choose from our wide selection of secondary antibodies that G Creci S mu are labeled with fluorescein FITC rhodamine TRITC Rhodamine Yellow 941 572 65 000 Texas Red a form of rhodamine R phycoerythrin or allophycocyanin fluorescent dyes Find an antibody with the specificity needed for nearly any immunofluorescence experiment R Phycoerythrin Yellow 480 545 565 578 2x 10 Texas Red Red 596 615 80 000 Allophycocyanin Red 620 645 660 7x 10 T Molar extinction coefficient M cm Spectral properties of Thermo Scientific DyLight Fluorescent Dyes Emission DyLight Dye Ex Em et Spectrally Similar Dyes Blue 405 400 420 30 000 Alexa Fluor 405 and Cascade Blue Dyes Green 488 493 518 70 000 Alexa Fluor 488 fluorescein and FITC Dyes Yellow 549 560 574 150 000 Alexa Fluor 546 Alexa Fluor 555 Cy3 and TRITC Dyes Red 594 593 618 80 000 Alexa Fluor 594 and Texas Red Dyes Red 633 638 658 170 000 Alexa Fluor 633 Dye Red 649 654 673 250 000 Alexa F
47. 93T cell lysate in lanes 1 5 of Panel C and 25 12 5 6 25 3 1 and 0 39 ug in lanes 1 5 of Panel D Protein detection was achieved as follows Panel A Mouse anti cyclin D1 antibody BD Pharmingen on PVDF Panel B Rabbit anti beta catenin antibody LabVision on PVDF Panel C Mouse anti GAPDH antibody Millipore on nitrocellulose Panel D Rabbit anti cdk2 antibody Upstate on PVDF Specialized Western Blotting Kits In additional to our traditional SuperSignal Western Blotting Substrates and kits we offer specialized kits for the detection of histidine tagged proteins phosphoproteins O Glc NAc post translational modifications multiple target proteins on a single Western blot and target proteins to verify siRNA Reagent gene knockdown Reach for Thermo Scientific Pierce Protein Detection Products for specificity sensitivity speed and convenience Thermo Scientific SuperSignal West Pico HisProbe Kit Specitic detection of histidine tagged fusion proteins This chemiluminescent system uses HisProbe HRP chemistry to overcome the limitations of anti histidine antibodies and other detection strategies HisProbe HRP is more specific for poly histidine tags reducing background problems Unlike anti His antibodies HisProbe HRP can recognize polyhistidine tags independent of adjacent tags Highlights e Specific more specific for the detection of histidine tagged fusion proteins than anti His antibodies Figure 8 e Fast
48. Detection Kit does not perform well with Bio Rad Ready Gel Precise Protein Gels or Gradipore iGel Gels studies showed 25 times lower sensitivity and require individual optimization e The recommended gel thickness for use with this kit is 0 75 1 5 mm e The recommended crosslinking of gel is 8 18 4 20 or 10 20 gradient When using Pierce In Gel Detection Technology with homemade gels the glass plates must be siliconized before pouring the gel Please visit our website to review the protocol and see other tips on optimizing the Pierce In Gel Detection Method Ordering Information Product Description Pkg Size 33500 Pierce In Gel Chemiluminescent Kit Detection Kit Rabbit Sufficient reagents to perform 10 mini gel detections Includes Pierce In Gel Substrate 110 ml Stabilized Goat Anti Rabbit HRP 10 pl Dilution Buffer 50 ml BupH Pack PBS Buffer 17 packs 10 Tween 20 5x 10 ml Incubation Colander 1 unit Pre cut Cellophane 10 sheets CL XPosure Film 5 x 7 25 sheets 33505 Pierce In Gel Chemiluminescent Kit Detection Kit Mouse Includes same components as Product 33500 except it contains Goat Anti Mouse HRP instead of Goat Anti Rabbit HRP 10 ul 33550 Pierce In Gel Detection 110 ml Chemiluminescent Substrate 33499 Incubation Colander 1 unit t See patent information on inside back cover References Desai S et al 2001 Anal Biochem 297 94 98 Desai S et al 2002 Immunodetection of proteins wit
49. P id is a trademark of Millipore Corporation Hoefer is a trademark of Hoefer Inc Contact Information Belgium and Europe the Middle East and Africa Distributors Tel 32 53 85 71 84 France Tel 0 800 50 82 15 The Netherlands Tel 076 50 31 880 Germany Tel 0228 9125650 United Kingdom Tel 0800 252 185 Switzerland Tel 0800 56 31 40 Email perbio euromarketing thermofisher com www thermo com perbio United States Tel 815 968 0747 or 800 874 3723 Customer Assistance E mail Pierce CS thermofisher com www thermo com pierce 2009 Thermo Fisher Scientific Inc All rights reserved These products are supplied for laboratory or manufacturing applications only Unless indicated otherwise on the inside back cover all trademarks are property of Thermo Fisher Scientific Inc and its subsidiaries Thermo SCIENTIFIC
50. Product 89888 e Precise Protein Gels many available see page 4 e Tris Hepes SDS Running Buffer Product 28398 e Lane Marker Reducing Sample Buffer 5X Product 39000 e Lane Marker Non Reducing Sample Buffer 5X Product 39001 s Pierce Blue Prestained Protein Molecular Weight Marker Product s 26681 and 26685 e Pierce Chemiluminescent Prestained Peroxidase labeled Protein Molecular Weight Marker Product 26651 e Pierce Prestained 3 Color Protein Molecular Weight Marker Product 26691 e DyLight Dual Labeled Fluorescent Marker Product 22859 and 26665 Electro Transfer l Transfer proteins to membrane e Fast Semi Dry Blotter Product 88217 e Methanol Free Transfer Buffer Product 35040 e Fast Semi Dry Transfer Buffer Product 35035 e Tris Glycine Transfer Buffer Product 28380 e Pierce Reversible Protein Stain Kit for Nitrocellulose Membranes Product 24580 and for PVDF Membranes Product 24585 e Pierce Western Blot Signal Enhancer Product 21050 e Pierce Antibody Extender NC Product 32110 and 32105 e Nitrocellulose Membrane 0 2 pm Product s 77012 88013 and 88024 e Nitrocellulose Membrane 0 45 um Product s 77010 77011 88014 and 88025 e PVDF Membrane 0 45 um Product s 88585 and 88518 e Low fluorescence PVDF Membrane 0 2 um Product 22860 e Western Blotting Filter Paper Product 88600 For detection of proteins that cannot be
51. RP 0 2 ug ml SuperSignal West Pico Substrate Product 34080 was used for detection of Cdk1 protein A B C D Clean Blot Clean Blot Detection Detection GAR HRP Reagent GAR HRP Reagent NF B Bax N xB KB N X x NXN X x N xB xB D LP A 5 RS x G A A G A A Figure 5 Reveal your target protein with Thermo Scientific Clean Blot Detection Reagent HRP To demonstrate unmasking of the target protein we performed IP and Western blot experiments NEB and Bax were immunoprecipitated from A549 lysate using Protein A G Agarose Resin and rabbit anti NFicB Panels A and B and rabbit anti Bax Panels C and D Panels A and C were detected with goat anti rabbit HRP which masked the target Panels B and D were detected with the Clean Blot Detection Reagent HRP revealing the target protein To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor Ordering Information Product Description Pkg Size 21230 Clean Blot IP Detection Reagent HRP 2 5 ml Sufficient reagent for approximately 100 Western blots 21232 Clean Blot IP Detection Kit HRP Kit Sufficient reagent for approximately 2 000 cm of membrane Clean Blot Detection Reagent HRP 2 5 ml StartingBlock T20 TBS Blocking Buffer 1L Pierce ECL Detection Reagent 1 125 ml Peroxide Solution Pierce ECL Detection Reagent 2 125 ml Luminol Enhancer Solution 21233 Clean Blot IP Detection Reagent AP 2 5 ml Suffi
52. ack ground noise than other popular chemiluminescent substrates for AP providing a better signal to noise ratio and a clearer image Because signal generation is immediate there s no need to wait 15 to 30 minutes for a measurable signal Figure 7 Lumi Phos Substrate provides high sensitivity and low background Serial dilutions of recombinant mouse IL 2 were separated electrophoretically on a 4 20 SDS polyacrylamide gel The separated protein was then transferred to nitrocellulose membrane followed by block ing The membranes were subsequently incubated in a 1 500 1 ug ml dilution of purified Rat Anti Mouse IL 2 followed by a 1 5 000 200 ng ml dilution of AP labeled Goat Anti Rat IgG The membranes were washed and then incubated in Lumi Phos WB Substrate for five minutes before film exposure Table 5 Thermo Scientific Substrates guide Highlights e High sensitivity able to detect 1 2 pg or 71 attomoles of the target ligand mouse IL 2 e Low background high signal to noise ratios produce clear blots e Inexpensive less expensive than other AP substrates based on 2007 U S list prices and there is no need to purchase additional enhancers for nitrocellulose membranes e Long signal duration allows you to redevelop blots over and over e Immediate strong signal no more waiting 15 to 30 minutes for the signal to become strong enough to detect e Ready to use no mixing required with this one com
53. anch office or distributor STEP STEP SDS PAGE Electro Trangrer Detection of Difficult to transfer Proteins The major reason that proteins are blotted or adsorbed onto a membrane for detection with an antibody is that the proteins on a membrane are more accessible to immunochemical reagents antibodies etc than are proteins within polyacrylamide gels A recent advance in the field of Western blotting involves immu nodetection of proteins directly in the gel This method Thermo Scientific Pierce In Gel Detection circumvents the transfer and blocking steps entirely enabling immunoblotting techniques to be applied to proteins that cannot be transferred efficiently from a gel to a membrane Because there s no transfer step no protein is lost in the process Figure 13 and no artifacts are introduced into the data This makes Pierce In Gel Detection an ideal control experi ment to confirm results obtained by Western blotting and to study proteins that cannot be transferred to a membrane Another feature of the Pierce In Gel System is that it does not require a blocking step eliminating the chance of cross reactivity with the blocking buffer This saves time because no blocking buffer optimization is necessary and background Is often lower than with traditional Western blotting STEP STEP Formul Wash Buff 1 2 3 4 5 6 7 8 9 10 11 12 13 4 Figure 13 Protein left in a gel after transfer to a nitrocell
54. anti Mouse HRP Product 31434 and SuperSignal West Dura Substrate Product 34075 The blot was exposed to film for 30 seconds resulting in considerable background speckling A The film was then treated with Pierce Background Eliminator for 2 minutes to eliminate the background speckling B STEP STEP Formul gt Blocki Wash Buff S Highlights e Reduces signal evenly over the film no altering of results e Fast easy background elimination from overexposed speckled or shaded films e Works with any X ray film new or old e No need for time consuming re exposures to find the optimal image e No need to re optimize assay reagents to obtain the optimal image Remove background from any application that uses X ray film exposures including e Western Northern and Southern blots that use SuperSignal Substrates and the Thermo Scientific Pierce Chemiluminescent Hybridization and Detection Kit e In gel detection systems e Gel shift assays e Ribonuclease protection assays RPA Ordering Information Product Description Pkg Size 21065 Pierce Background Eliminator Kit Sufficient reagent to prepare 3 L of working solution Includes Pierce Reagent A 100 ml Pierce Reagent B 100 ml For more information or to download product instructions visit www thermo com pierce Blotting with Chemiluminescence Most of the time troubleshooting a problem with any given Western blot system involves
55. as By combining 24 hour light emission with ultraintensity SuperSignal West Dura Substrate allows researchers to take full advantage of all the features offered by imaging instruments SuperSignal West Dura Substrate provides the maximum light duration allowing multiple extended exposures We performed an experiment to compare SuperSignal West Dura Substrate with GE Healthcare Amersham ECL Plus Substrate using the manufacturers protocols Recombinant mouse IL 2 0 003 50 ng was applied to a polyacrylamide gel and electrophoresed The proteins were trans ferred to PVDF for the GE Healthcare ECL Plus Substrate and to nitrocellulose for the SuperSignal Substrate The primary antibody for both substrates was used at a 1 pg ml The secondary antibodies were used at 10 ng ml for SuperSignal West Dura Substrate and 20 ng ml for GE s ECL Plus Substrate A five minute film exposure produced a high signal to noise ratio for the SuperSignal West Dura System with detection down to 3 pg Figure 5A but produced high background for the ECL Plus Substrate and detection down to only 800 pg Figure 5C A 30 min ute exposure at F1 6 on the CCD camera demonstrated detection down to 12 5 pg with the SuperSignal Product Figure 5B When the GE Healthcare ECL Plus Blot was exposed to the CCD camera at F1 6 the exposure was stopped at 15 minutes because of the intense background Signal was difficult to distinguish above background Figure 5D Highlights
56. ate dilutions see the table below The necessary dilution will vary depending on the enzyme conjugate used the primary antibody used in Step 6 and the amount of antigen that was transferred Recommended Secondary Antibody Dilutions from 1 mg ml stock 1 1 000 1 15 000 or 1 0 067 ug ml SuperSignal 1 20 000 1 100 000 or 10 50 ng ml West Pico Substrate SuperSignal 1 100 000 1 500 000 or 2 0 10 ng ml West Femto Substrate SuperSignal 1 50 000 1 250 000 or 4 0 20 ng ml West Dura Substrate Lumi Phos WB Substrate 9 1 5 000 1 25 000 or 40 200 ng ml Repeat Step 7 to wash away any unbound enzyme conjugated secondary antibody It is crucial to thoroughly wash the membrane after the incubation with the enzyme conjugate If the working solution has not been prepared prepare it now For Supersignal West Substrates mix equal volumes of the Luminol Enhancer Solution and the Stable Peroxide Solution Prepare a sufficient volume to ensure that the blot is completely wetted with substrate and the blot does not dry out Lumi Phos WB Substrate is provided in a ready to use format but it should be brought to room temperature Recommended volume 0 1 ml cm of blot surface To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor Incubate the blot with SuperSignal Substrate Working Solution for 5 minutes or with Lumi Phos WB Substrate Working Solution for 3 minutes
57. ately 20 ml for an 8 x 10 cm blot Alternatively the blot can be incubated with a solution of 2 w v SDS 62 5 mM TriseHCl 100 mM 2 mercaptoethanol pH 6 8 for 30 90 minutes at 50 70 C However these reaction conditions are much harsher than Restore Western Blot Stripping Buffer and are more likely to interfere with future ligand antibody interactions Note In general high affinity antibodies will require at least 15 minutes of stripping and may require an incubation temperature of 37 C Alternatively use Restore PLUS Stripping Buffer which is optimized for high affinity antibodies 2 Remove the blot from the Restore Western Blot Stripping Buffer and wash in Wash Buffer 3 Test for the removal of the immunodetection reagents A To test for complete removal of the HRP label incubate the membrane with SuperSignal West Working Solution and expose to film If no signal is detected with a 5 minute exposure the HRP conjugate has been successfully removed from the antigen or primary antibody B To test for complete removal of the primary antibody incubate the membrane with the HRP labeled secondary antibody fol lowed by a wash in wash buffer Apply SuperSignal West Working Solution If no signal is detected with a 5 minute exposure the primary antibody has been successfully removed from the antigen C If signal is detected with experiment A or B place the blot back into Restore Western Blot Stripping Buffer for an additiona
58. ble 1 Cost comparison of 5 x 7 sheets Product Cost per sheet U S Price Thermo Scientific CL XPosure Film Blue X ray Film 1 01 Kodak X Omat Blue XB Film Blue X ray Film Perkin Elmer 2 91 Kodak BioMax MR 1 Gray X ray Film GE Healthcare 5 20 Source 2008 Online Catalogs Ordering Information Product Description Pkg Size 34090 CL XPosure Film 5 x 7 in 13 x 18 cm 100 pkg 34092 CL XPosure Film 5 x 7 in 13 x 18 cm 25 pkg 34089 CL XPosure Film 7 x 9 5 in 18 x 24 cm 100 pkg 34091 CL XPosure Film 8 x 10 in 20 x 25 cm 100 pkg 34093 CL XPosure Film 8 x 10 in 20 x 25 cm 50 pkg For more information or to download product instructions visit www thermo com pierce Enzyme Substrates Optimize the Signal to Noise Ratio Signal to noise ratio S N ratio refers to how much relevant content signal something has as opposed to non relevant content noise The term is from the radio industry but is often applied to Western blotting In Western blotting the signal is the density of the specific protein band being probed for the noise is the density of the background Optimizing the S N ratio is often more important than increasing the sensitivity of the system The sensitivity of the system is irrelevant if the signal cannot be distinguished from the noise The General Troubleshooting Guide in the next section contains many tips on optimizing the S N including a method of increasing the signa
59. brane Detailed procedures for detection of a Western blot vary widely One common variation involves direct vs indirect detection Figure 1 With the direct detection method the primary antibody that is used to detect an antigen on the blot is labeled with an enzyme or fluorescent dye This detection method is not widely used as most researchers prefer the indirect detection method for a variety of reasons Table 1 In the indirect detection method a primary antibody is added first to bind to the antigen This is followed by a labeled secondary antibody that is directed against the primary antibody Labels include biotin fluorescent probes such as fluorescein or rhodamine and enzyme conjugates such as horseradish peroxidase or alkaline phosphatase The indirect method offers many advantages over the direct method Table 2 Substrate Detectable 34 Product Enzyme Substrate Detectable a4 Product Enzyme 1A Direct Detection 1B Indirect Detection Figure 1A In the direct detection method labeled primary antibody binds to antigen on the membrane and reacts with substrate creating a detectable signal 1B In the indirect detection method unlabeled primary antibody binds to the antigen Then a labeled secondary antibody binds to the primary antibody and reacts with the substrate Table 1 Direct detection method Advantages Disadvantages e t is a quick methodology because only one antibody i
60. capital investment for instrument Prices except SNAP id Blot Holder based on Thermo Scientific Pierce Western Blotting Products For more information or to download product instructions visit www thermo com pierce Ordering Information Product Description Pkg Size 35050 Pierce Fast Western Blot Kit ECL Substrate Kit Sufficient reagents for 25 Western blots 8 x 10 cm probed with mouse or rabbit antibody Includes Antibody Diluent 500 ml 10X Wash Buffer 250 ml Optimized HRP Reagent 25 ml Pierce ECL Detection Reagent 1 125 ml Pierce ECL Detection Reagent 2 125 ml 35055 Pierce Fast Western Blot Kit ECL Substrate Ki Sufficient reagents for 5 Western blots 8 x 10 cm probed with mouse or rabbit primary antibody t Includes Antibody Diluent 100 ml 10X Wash Buffer 50 ml Optimized HRP Reagent 5 ml Pierce ECL Detection Reagent 1 25 ml Pierce ECL Detection Reagent 2 25 ml A Classical Fast Western Blot 1 2 3 4 12 3 4 B 12 3 4 1 2 3 4 12 3 4 5 12 3 4 5 12 3 4 5 12 3 4 5 Obtain comparable results to the Classic Western blotting protocol using Thermo Scientific Pierce Fast Western Blot Kit ECL Substrate The detection sensitivity for various target proteins was compared using the Fast Western Blot Kit and the classical Western blot protocol Panels A and B contained 10 2 0 4 and 0 08 ug of protein from A549 cell lysates in lanes 1 4 respectively Panels C and D contained 2 1 0 5 0 25 and 0 125 ug of 2
61. cient reagent for approximately 100 Western blots t See patent information on inside back cover To view data on our Clean Blot Detection Reagents visit www thermo com pierce 29 STEP STEP R LS B A fr m D gt i PA 14 E Electro Transfer Thermo Scientific Antibody Binding Proteins Protein A Binds specifically to the Fe region of immunoglobulin molecules especially IgG Highlights e Isolated from native Staphylococcus aureus MW 42K e Contains four IgG binding sites Ordering Information Product Description Pkg Size 21181 Protein A 5 mg 29989 Biotinylated Protein A 1mg Protein A Recombinant No enterotoxins present as there may be from Staphylococcus derived Protein A Highlights e Harvested from a nonpathogenic form of Bacillus which has been genetically designed to manufacture and secrete carboxy terminus truncated MW 44 6K recombinant Protein A Ordering Information Product Description Pkg Size 21184 Purified Protein A 5 mg 32400 Pierce Purified Recombinant 1 mg Protein A Peroxidase Conjugated Protein G Recombinant Useful for a variety of immunological and biochemical techniques Highlights e Protein G is a bacterial cell wall protein isolated from group G Streptococci MW 22K e Binds to most mammalian immunoglobulins through their Fe regions e Albumin and cell surface binding sites have been removed from this recombinant form to reduce nonspecific binding wh
62. d BSA pg Figure 7 Densitometry data on dot blot comparing before and after use of the Thermo Scientific Pierce Background Eliminator Dot blots were prepared on nitrocellulose Product 77010 using Biotinylated BSA Product 29130 at 1 000 250 62 5 and 15 6 pg The blot was blocked with SuperBlock Blocking Buffer in PBS Product 37515 and incubated with a 1 50 000 dilution of SA HRP Product 21126 The blot was then washed for 30 minutes incubated in SuperSignal West Pico Substrate Product 34080 and exposed to film Product 34092 for 5 minutes The resulting film had high background that was Cut into four strips each containing three replicates per concentration The Background Eliminator Working Solution was used on separate film strips at 1 2 5 and 4 minutes leaving a control strip for comparison After scanning on a densitometer the relative signal intensity was compared The signal intensity decreased evenly with time when treated with the Background Eliminator Solution maintaining similar slopes on a dose response curve After Using Thermo Scientific Pierce Solution Before Using Thermo Scientific Pierce Solution 4 A B Figure 8 Thermo Scientific Pierce Background Eliminator erases speck ling Recombinant Human TNFo was electrophoresed on a 4 20 SDS polyacrylamide gel and transferred to a nitrocellulose membrane The mem brane was blocked and detected with Mouse anti Human TNFo followed by Goat
63. d saline TBS or phosphate buffered saline PBS without any additives More commonly a detergent such as 0 05 Tween 20 Detergent Product 28320 is added to the buffer to help remove nonspecifi cally bound material Another common technique is to use a dilute solution of the blocking buffer along with some added detergent to help minimize background For best results use high purity deter gents such as Surfact Amps Detergents for Western blotting Thermo Scientific BupH Dry Buffers The most advanced versatile time saving buffer products available The ultimate in convenience 1 Reach for the sealed foil pack stored conveniently on the bench top 2 Open pour into beaker and add water 3 The fresh buffer is ready to use in practical amounts so there s no waste The ultimate in versatility 1 Routine buffers are designed for use in Western blotting dialysis crosslinking ELISAs immunohistochemistry protein plate coating biotinylation and other applications 2 Using one buffer source maintains consistency and minimizes variables The ultimate in integrity 1 BupH Buffers are protected from contamination and are fresh every time 2 Perform applications with confidence in quality buffers 3 Test assured with our commitment to quality management standards The ultimate in time savings 1 Making routine buffers is no longer time consuming 2 No component measurement pH adjustment quality validation
64. detection limits Figure 6 that s zeptomole level detection e Economical conserve precious antibodies with up to 1 100 000 primary antibody dilutions and 1 500 000 secondary antibodies dilutions e Intense releases the most intense signal generated by chemiluminescent systems making it easy to capture an image on film or via an imager system e Quantitative over two orders of magnitude Feissner R et al 2003 Anal Biochem 315 90 94 Lower detection limit e Low femtogram 10 e Mid zeptomole 10 Signal duration e 8 hours Suggested antibody dilutions from 1 mg ml stock e Primary 1 5 000 1 100 000 e Secondary 1 100 000 1 500 000 Reagent stability e 1 year at 4 C or 6 months at RT Table 4 A comparison of Thermo Scientific Chemiluminescent Substrates SuperSignal West Pico Chemiluminescent Substrate Pierce ECL Substrate Primary Benefit e The same signal intensity at half the price of competing ECL Substrates e Low microgram 10 e High picomoles 10 Lower Detection Limit Signal Duration e 30 minutes 2 hours e 6 8 hours Suggested Antibody Dilutions e Primary 1 100 1 5 000 Room Temperature RT s 1 hour e 24 hours Working Solution Stability Stock Solution Shelf Life 1 year at 4 C e 1 year at RT e Twice the signal for about half the price of competing products e Low picogram 10 e Mid attomole 10 e Primary 1 1 000 1 5 000 e Secondary 1 1 000 1 15
65. ding your valuable research time and money looking for the right antibody use our antibodies with confidence We have done all the work to offer you the best intracellular target antibodies available Supplier A Supplier B Thermo Scientific 1 2 3 1 2 3 1 2 3 Figure 1 Comparison of anti AKT2 antibody specificity using siRNA mediated protein knockdown MCF7 cells were transfected with Thermo Scientific Dharmacon ON TARGET plus SMARTpool AKT2 siRNA Reagent Cell lysates were analyzed by Western blot Two other suppliers antibodies were compared to the Thermo Scientific Anti AKT2 Antibody Product 82311 The AKT2 band upper band detected by the Thermo Scientific Antibody which is knocked down by AKT2 siRNA is not recognized by the two other suppliers ATK2 antibodies Lane 1 Mock transfection Lane 2 Control pool siRNA and Lane 3 AKT2 siRNA The arrow indicates the 60 kDa AKT2 protein band 220 100 20 Supplier A Thermo Scientific Figure 2 Comparison of antibodies for detecting CDK9 protein by Western blot A549 and HeLa cell lysates lanes 1 and 2 respectively were analyzed by Western blot using Thermo Scientific Anti CDK9 Antibody Product 82340 and another supplier s antibody After many optimization experiments the supplier s antibody did not detect a definitive protein band The arrow indicates the 45 kDa CDK9 protein 1 2 1 2 For more information or to download product instructions visit www th
66. dy 1 500 Secondary Antibody 1 5 000 Primary Antibody 1 5 000 Secondary Antibody 1 50 000 Figure 3 Example of signal intensity on a Western blot using Thermo Scientific SuperSignal West Dura Substrate and antibodies at various concen trations Blots were optimized with SuperSignal West Dura Chemiluminescent Substrate Blot 1 primary and secondary antibody concentrations are too high The bands are too intense and blur together resulting in poor resolution A large number of nonspecific bands are also visible To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor Electro Transfer Dot Blot Protocol for Optimization of Antigen and Antibody Concentrations The optimal antibody concentrations to use with a given antigen are dependent on the antigen and antibody themselves The affinity avidity of the antibody for the antigen and the specific activity of both the primary and secondary antibody will vary The optimal antigen and antibody concentrations can be determined by performing complete Western blots with varying concentrations of antigen and antibody Alternatively a faster and easier method is to perform a dot blot procedure The following is a dot blot protocol using SuperSignal West Pico Substrate When using other Thermo Scientific Substrates refer to the product instruc tions for recommended antigen antibody concentrations Note All antibody dilutions a
67. dy conjugates are affinity purified to minimize cross reactivity e Superior photostability e pH insensitive pH 4 9 e High water solubility e Compatible with common fluorescence instrumentation Spectrally Ex Em et Similar Dyes 400 420 30 000 Alexa Fluor 405 and Cascade Blue Dyes 493 518 70 000 Alexa Fluor 488 fluorescein and FITC Dyes 560 574 150 000 Alexa Fluor 546 Alexa Fluor 555 Cy3 and TRITC Dyes 593 618 80 000 Alexa Fluor 594 and Texas Red Dyes 638 658 170 000 Alexa Fluor 633 Dye 654 673 250 000 Alexa Fluor 647 and Cy5 Dyes 692 712 140 000 Alexa Fluor 680 and Cy5 5 Dyes 752 778 220 000 Alexa Fluor 750 and Cy7 Dyes 777 790 270 000 IRDye 800 Dye Ordering Information Conjugates Package size for these items is 1 mg at 1 mg ml Product DyLight DyLight DyLight DyLight DyLight DyLight DyLight DyLight DyLight Description 405 Dye 488 Dye 549 Dye 594 Dye 633 Dye 649 Dye 680 Dye 750 Dye 800 Dye Goat Anti Mouse IgG H L 35502 35507 35515 35518 35521 Goat Anti Mouse IgG 35500 35503 35508 35511 35513 35516 35519 Highly Cross Adsorbed Goat Anti Rabbit IgG H L 35552 35557 35565 35568 35571 Goat Anti Rabbit IgG 35550 35553 35558 35561 35563 35566 35569 Highly Cross Adsorbed Streptavidin 21832 21837 21845 21848 21851 NeutrAvidin Biotin Binding 22832 22837 22845 22848 22853 Protein For more information or to download product instructions visit www thermo com pierce Enzyme Substrates Thermo Scien
68. ection of per oxidase conjugated antibodies on immunoblots J Virol Meth 24 221 235 For more information or to download product instructions visit www thermo com pierce Antibody Production Technical Handbook This 69 page handbook helps you choose the best methods to produce purify fragment and label antibodies Topics include basic immunology carrier proteins adjuvants antibody purification methods antibody frag mentation with proteases and label ing antibodies with a variety of tags e g biotin fluorophores enzymes iodine for purification or detection Assay Development Technical Handbook This 74 page guide features protocols and products that can improve your ELISAs Featured products include coated plates protein standards blockers buffers secondary antibodies and substrates Fluorescent Products Guide This 16 page brochure features Thermo Scientific DyLight Dyes and Conjugates Dye Removal Columns Antibody Labeling Kits Western Blotting Kits and MW Markers Protein Purification Technical Handbook This 81 page handbook provides protocols and technical and product information to help maximize results for protein purification It also includes background and trouble shooting advice for covalent coupling of affinity ligands to chromatography supports avidin biotin binding affinity purifica tion of antibodies IP and co IP affinity procedures for contaminant removal a
69. ectors and exposed to CL XPosure Film Product 34090 for 90 seconds Thermo Scientific Pierce ECL Reagent GE Healthcare Protein per well 100 120 kDa Amersham ECL Reagent Protein per well 100 120 kDa MW 450 225 113 56 28 ng MW 450 225 113 56 28 ng Marker Marker 5 minute exposure 5 minute exposure Thermo Scientific Pierce ECL Substrate Western blot detection of B galactosidase expressed from Escherichia coli lysate Dilutions of E coli cell lysate were prepared and separated by electrophoresis The proteins were transferred to PVDF membranes Product 88585 Membranes were blocked with 5 skim milk and then incubated with Mouse Anti B galactosidase AB 1 Lab Vision Fremont CA at 1 pg ml The membranes were washed and then incubated with 0 2 ug ml of HRP conjugated Goat Anti Mouse IgG Product 31430 and then washed again Working solutions of the substrates were prepared according to the manufacturers instructions and added to the mem branes for 1 minute The membranes were placed in plastic sheet protectors and exposed to CL XPosure Film Product 34090 for five minutes Thermo Scientific Pierce ECL Reagent GE Healthcare Amersham ECL Reagent Protein per well 20 kDa Protein per well 20 kDa MW 500 250 125 63 31 pg MW 500 250 125 63 21 pg Marker Marker 1 minute exposure 1 minute exposure Thermo Scientific Pierce ECL Substrate Western blot detection of recombinant bovine TNF a Dilutions
70. efore the conjugation process Selected Pierce Antibodies have been further purified to minimize cross reactivities to other species serum proteins and Is indicated by min x Species Sr Prot The key to abbreviations for the individual species is shown in Table 3 Pierce Polyclonal Conjugated Antibodies contain bovine serum albumin as a stabilizer Table 4 lists the typical conjugate working dilutions for ELISAs immunoblotting and immuno histochemical techniques Table 4 Typical dilution ranges for Thermo Scientific Pierce Polyclonal Conjugated Antibodies Conjugate ELISA AP 1 5 000 1 50 000 Immunoblotting Immunohistochemistry 1 2 500 1 25 000 1 500 1 5 000 Peroxidase 1 5 000 1 200 000 1 25 000 1 500 000 1 500 1 5 000 for SuperSignal for SuperSignal ELISA Products West Products Fluorescein 1 50 1 200 Rhodamine 1 50 1 200 DyLight Dyes 1 100 1 500 1 10 000 1 75 000 1 1 000 1 5000 For more information or to download product instructions visit www thermo com pierce Enzyme Substrates Stabilized HRP Conjugates Pre diluted stable solutions of our most popular secondary antibodies Stabilized HRP Conjugates are secondary antibody conjugates with horseradish peroxidase HRP enzyme that are stabilized in pre diluted form for greater accuracy and convenience in preparing working solutions Thermo Scientific Stabilized HRP Conjugates are accurately prepared dispensed and supplied a
71. en Protein G is used to purify identify or locate immunoglobulins e Useful for separating albumin from crude human or mouse IgG samples e Binds with greater affinity to most mammalian immunoglobulins than Protein A including human IgG and rat IgG e Does not bind to human IgM IgD and IgA STEP STEP Formul Wash Buff Ordering Information Product Description Pkg Size 21193 Pierce Purified Recombinant Protein G 5 mg 29988 Biotinylated Protein G 0 5 mg 31499 Protein G Peroxidase Conjugated 0 5 mg Protein A G Recombinant Produced by gene fusion of the Fc binding domains of Protein A and Protein G Highlights e Protein A G is a 50 449 dalton protein containing 442 amino acids 43 of which are lysines e Binds well to immunoglobulins over a broad pH range pH 4 9 e Contains four Protein A Fc binding domains and two Protein G Fe binding domains e Binds all IgG subclasses of mouse immunoglobulins making it an excellent tool for purification and detection of mouse monoclonal antibodies Ordering Information Product Description Pkg Size 21186 Pierce Purified Recombinant Protein A G 5 mg 32391 Protein A G Alkaline 0 5 mg Phosphatase Conjugated 32490 Protein A G Peroxidase Conjugated 0 5 mg Protein L Recombinant Binds a wider range of Ig classes and subclasses including all classes of IgG and single chain variable ScFv and Fab fragments Highlights e Protein L is an immunoglobulin binding protei
72. ent antibodies Western blots of HeLa cell lysate protein diluted 750 83 3 ng were detected with SuperSignal West Dura Chemiluminescent Substrate The first blot used polyclonal rabbit anti JAK 1 primary antibody BD PharMingen San Jose CA at 1 2 000 dilution with an HRP secondary conjugate diluted at 1 350 000 The same blot was stripped for 5 minutes at room temperature in Restore Western Blot Stripping Buffer and then re probed with purified Mouse Anti Human Bak monoclonal primary antibody at 1 1 000 with the HRP secondary conjugate at 1 100 000 Five percent nonfat milk with 0 05 Tween 20 was used for blocking To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor STEP STEP SDS PAGE Electro Trangrer Thermo Scientific Restore PLUS Western Blot Stripping Buffer A new formulation for high affinity antibodies that require special treatment When researchers require a robust but gentle Western blotting stripping buffer the original Restore Western Blot Stripping Buffer has been the buffer of choice However some antibodies remain difficult to remove from Western blots and require longer incuba tion times or incubation temperatures greater than 22 C Restore PLUS Western Blot Stripping Buffer was developed to reduce incubation times while keeping incubations at room temperature High affinity antibodies can be quickly and effectively stripped from Western blot
73. equired The process can be halted when the signal is clearly visible and the background is at a minimum thereby increasing the S N ratio without altering the data s integrity Figure 5 Pierce Background Eliminator provides fast easy removal of background image on exposed X ray film for Western Northern or Southern blots so you can see your results clearly High background shading overexposed bands and speckling are problems inherent to film exposure High background and shading can be caused by overexposure poor use of blocking buffer or inappropriate enzyme labeled probe or antibody concen tration Overexposed bands are a common occurrence when the enzyme labeled probe or antibody used is too concentrated or if the film was exposed for too long Speckling and shading occur when enzyme conjugates form complexes and precipitate on the blot The Pierce Background Elimination Kit can correct all these problems without the need to re expose your blot to film or re do the experiment allowing you to visualize your data within minutes Figures 6 8 The Pierce Solution can be used with newly exposed films or exposed films that have been stored for years In addition the Pierce Kit can be used with any brand of film For applications requiring densitometric measurement the Pierce Background Eliminator reduces signal evenly over the film so that relative densitometry values are consistent Figure 7 The procedure is simple Immerse your
74. er e Optimize blocking time and or temperature Block for at least 1 hour at RT or overnight at 4 C e Add Tween 20 Detergent to blocking buffer Use a final concentration of 0 05 Tween 20 Detergent Skip this step if you use StartingBlock T20 Blocking Buffer in PBS Product 37539 or TBS Product 37543 or SuperBlock T20 Blocking Buffer in PBS Product 37516 or TBS Product 37536 These buffers already contain Tween 20 Detergent at optimized concentrations e Make up antibody dilutions in blocking buffer with 0 05 Tween 20 Detergent Cross reactivity of antibody with e Use a different blocking buffer other proteins in blocking buffer e Do not use milk with avidin biotin systems Milk contains biotin e Test for cross reactivity Block a clean piece of membrane incubate with antibodies and then detect with SuperSignal Chemiluminescent Substrate e Reduce the concentration of the HRP conjugate Membrane was not wetted properly Wet membrane according to the manufacturer s instructions e Do not handle membrane with bare hands Always wear clean gloves or use forceps e Use a new membrane e Make sure the membrane Is covered with a sufficient amount of liquid at all times to prevent it from drying e Use agitation during all incubations e Incubate membranes separately to ensure that membrane strips are not covering one another during incubations e Handle membranes carefully damage to the membrane can cause nonspecific bindin
75. er is provided as a 10X concentrate it is also stable at a 1X concentration Ordering Information Product Description Pkg Size 34062 Pierce Stable Peroxide Buffer 10X 100 ml Substrates for HRP TMB with a molecular weight of 240 4 is most often used as a substrate for HRP in ELISAs However in the presence of HRP and peroxide a water soluble blue product is generated that can be precipitated onto a membrane Pierce TMB Blotting Product 34018 is a single component peroxidase substrate for Western blotting and immunohistochemistry Precipitating the product results in dark blue bands where the enzyme is located Pierce TMB Blotting is well suited to applications that require a high signal to noise ratio Ordering Information Product Description Pkg Size 34018 Pierce TMB Blotting 250 ml 4 CN has a molecular weight of 178 6 and can be used for chromogenic detection of HRP in blotting and histochemistry This precipitate is not as sensitive or as stable as TMB and DAB but the alcohol soluble precipitate photographs well and has a distinct blue purple color that can be useful in double staining applications Ordering Information Product Description Pkg Size 34012 Pierce CN 250 ml 34010 Pierce 4 Chloro 1 Napthol Powder 25 g powder 34011 Pierce 4 Chloro 1 Napthol Tablets 50 tablets 30 mg tablet For more information or to download product instructions visit www thermo com pierce DAB has a
76. erce Streptavidin Goat anti Mouse and Goat anti Rabbit Poly HRP are compatible with chromogenic fluorogenic and chemiluminescent HRP substrates used in ELISA Western blotting immunohisto chemistry IHC and nucleic acid hybridization assays Ordering Information Product Description Pkg Size 21140 Pierce Streptavidin Poly HRP 0 5 ml 0 5 mg ml 32260 Pierce Goat Anti Rabbit Poly HRP 0 5 ml 0 5 mg ml 32230 Pierce Goat Anti Mouse Poly HRP 0 5 ml 0 5 mg ml Storing Enzyme Conjugates We provide a variety of reagents to help preserve enzyme con jugate activity Typically conjugates are aliquoted in 50 100 ul increments using purified ethylene glycol Product 29810 as a preservative for 20 C storage Conjugates can maintain activity for up to two years An alternative to aliquoting is to use Pierce Peroxidase Conjugate Stabilizer Product 31503 diluting the conjugate 1 1 in the stabilizer and storing at 20 C for up to one year as a stock solution Pierce Peroxidase Stabilizer Diluent Product s 37548 and 37552 allow peroxidase conjugates to be reconstituted and stored at 4 C as a 1 1 000 or a 1 100 000 dilution Conjugate Stabilizers Ordering Information Product Description Pkg Size 37548 Pierce Peroxidase 200 ml Conjugate Stabilizer Diluent SD 37552 Pierce Peroxidase 1L Conjugate Stabilizer Diluent SD 31503 Pierce Peroxidase 25 ml Conjugate Stabilizer 29810 Ethylene Glycol 200 ml 50 aqueo
77. ermo com pierce Enzyme Substrates Thermo Scientific Pierce Antibody Catalog With the addition of ABR Affinity BioReagents to the Thermo Scientific family of products you Ordering Information Product Description See Below Fach package contains sufficient antibody for 10 mini blots Target Product Target Product Target Product a L now have access to more than ani aik me agi iii Beate 35 000 antibodies In 42 research AKT2 82311 CHEKI 82345 MDM2 82383 argas Inis book contains a nn ooo S S sampling of these antibodies For a aE CHEK p full list visit www thermo com abr ARF6 82313 CHUK 82347 MYC 82385 ATM 82314 CSNK2A1 82348 NCK1 82386 ATR 82315 CTNNB1 82349 NFKB1 82387 AURKB 82316 E2F1 82354 PUP 82388 Table 1 Variable performance of commercially available antibodies Results BAD 82317 EGFR 82355 PKR 82389 are listed as the percent of total antibodies tested BAX 82318 EP300 82356 PLCG1 82390 Result Total BCL2 82319 ERBB2 82357 PLK1 82391 No band detected 24 BCL2L1 82320 FOXO1A 82358 PPP2CA 82392 High background many nonspecific bands detected 26 BID 82321 FRAP1 82359 PRKACA 82393 Incorrect band as indicated by lack of siRNA knockdown 7 BIRC4 82322 GRB2 82360 PRKCA 82394 Band detected at appropriate MW 43 BIRC5 82323 GSK3A amp B 82361 PRKDC 82395 BRCA1 82324 GSK3B 82362 PTK2 82396 BUB1B 82325 HDAC1 82363 RAF1 82397 CASP3 82326 HDAC2 82364 RB1 82398 CASP8 82327 HDAC3 82365 RELA 82399 CASP9
78. exposed film in Pierce Background Eliminator Working Solution watch for desired image and stop the reaction by rinsing the film in water The Pierce Solution works quickly with ideal signal level typically attained in just a few minutes A Before using Thermo Scientific Pierce Background Eliminator B After using Thermo Scientific Pierce Background Eliminator Figure 5 Thermo Scientific Pierce Background Eliminator lightens overex posed bands Recombinant human wild type p53 baculovirus lysate was sepa rated on a 12 SDS polyacrylamide gel The proteins were transferred to a nitrocellulose membrane and blocked with SuperBlock Blocking Buffer in PBS Product 37515 The protein was detected with mouse anti p53 followed by Goat anti Mouse HRP Product 31434 and SuperSignal West Pico Substrate Product 34080 The membrane was exposed to film for 1 minute A The film had overexposed bands and was treated with Pierce Background Eliminator for 6 minutes The resulting image provided better visualization of the different p53 protein bands B A Overexposed Film L Old option Start over and re optimize antibody concentration and blocking buffer New option Use Thermo Scientific Pierce Background Eliminator C Four minutes later B Two days later Figure 6 Thermo Scientific Pierce Background Eliminator lightens the entire film evenly in four minutes vs the two days traditional methods require to start o
79. fic MW values are provided with each package Highlights e Colorimetric and chemiluminescent detect on membrane or in gel e Visual detection in gel already prestained does not require staining to detect in gel e Self contained peroxidase activity does not require an HRP antibody conjugate for chemiluminescence e Compatible with streptavidin HRP conjugates e Room temperature stable e Convenient packaging single dose in 48 well microtube plate Key consideration when using the Pierce Chemiluminescent Marker The peroxidase activity associated with the Pierce Chemiluminescent Marker is enzymatic Avoid denaturing or deactivating conditions to preserve activity Heating the gel during electrophoresis pH extremes denaturing agents strong reducing agents oxidizing agents and chelating agents will attenuate or quench peroxidase activity Ordering Information Product Description Pkg Size 26651 Pierce Chemiluminescent 1 x 48 Prestained Peroxidase Labeled microtube Protein Molecular Weight Marker Mix plate For more information or to download product instructions visit www thermo com pierce Enzyme Substrates After electrophoresis the protein must be transferred from the gel to a membrane There are a variety of methods that have been used for this process including diffusion transfer capillary transfer heat accelerated convectional transfer vacuum blotting transfer and electroelution he
80. g Contamination in buffers e Use new buffers e Filter buffers before use Contaminated equipment e Make sure electrophoresis equipment blotting equipment and incubation trays are clean and free of foreign contaminants e Make sure there are no pieces of gel left on the membrane after transfer Proteins can stick to the pieces of gel and cause background To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor STEP STEP STEP STEP Formul SDS PAGE Electro Trangrer Wash Buff oignal Possible Causes Precautions Solutions Proteins did not transfer properly e After transfer is complete stain the gel with a total protein stain to determine transfer efficiency to the membrane Note Total protein stains may not be able to detect low quantities of antigen e Use Thermo Scientific Pierce Reversible Membrane Stain to check membrane for transfer efficiency e Make sure there is sufficient contact between the gel and membrane during transfer e Make sure the transfer sandwich is assembled correctly e Be sure to follow the membrane manufacturer s instructions for wetting the membrane e Make sure transfer unit does not overheat during electroblotting procedure e Use positive control and or molecular weight markers e Optimize transfer time and current e Use Pierce Lane Marker Sample Buffer The tracking dye transfers to the membrane e Make sure sample
81. g Stripping Buffer HRP AP or biotin Product s 46428 46430 and 46431 for High Affinity e Clean Blot IP Detection Reagents HRPAP Antibodies e DyLight Secondary Antibody and Streptavidin Conjugates e IgG Elution Buffer Product s 21004 and 21009 Photostable and inexpensive alternatives to CyDye Fluors GE and Alexa Fluor Dye Invitrogen t See patent information on inside back cover To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor STEP STEP SDS PAG Electro Trangrer Thermo Scientific Precise Protein Gels Long shelf life short run time Thermo Scientific Precise Protein Gels are cast in a durable plastic cassette using a neutral pH buffer that prevents polyacrylamide breakdown and results in a long shelf life High resolution staining and transfer of proteins is accom plished quickly on these 1 mm thick gels Gels are individually packaged in an easy to open plastic pouch and are ready to run with no comb or tape to remove The gels are available in both gradient and fixed concentrations and in 10 12 and 15 well formats Highlights e 12 month guarantee ensures consistent performance e 45 minute run time provides results quickly e Sample wells hold up to twice the volume of Novex Brand gels 10 well 50 ul 12 well 30 pl 15 well 25 ul e Unique running buffer produces excellent separation and high resolution pro
82. g with chemiluminescence is still captured on film Often it is necessary to expose several films for different time periods to obtain the proper balance between signal and background The goal is to time the exposure of the membranes to the film so that the desired signal is clearly visible while the background remains low This is difficult to accomplish because the process cannot be observed and stopped when the desired endpoint is reached If the film is not exposed long enough underexposed the signal will not be visible If the film is exposed too long overexposed the signal may be lost in the background or separate bands may become blurred together An overexposed film can be fixed by incubating itin Pierce Background Eliminator Solution Product 21065 which effectively decreases the background without altering the integrity of the data This is done at the lab bench while watching the film and the process can be halted when the signal is clearly visible and background is at a minimum For more information on this method see page 57 Most instrument companies know and recommend SuperSignal West Substrates over other chemiluminescent substrates for use in their instruments Troubleshooting tips for chemiluminescence and cooled CCD cameras e SuperSignal West Dura and SuperSignal West Femto Substrates are the recommended substrates for use in imaging instruments e SuperSignal West Pico Substrate will work in imaging instr
83. gated Anti Rabbit IgG Anti Mouse IgG or NeutrAvidin Biotin Binding Protein e SuperBlock Blocking Buffer e TBS Wash Buffer e SuperSignal West Pico Substrate Ordering Information Product Description Pkg Size Standard Detection Kits 34082 SuperSignal West Pico Kit Mouse IgG Detection Kit 34083 SuperSignal West Pico Kit Rabbit IgG Detection Kit 34085 SuperSignal West Pico Kit Biotinylated Protein Detection Kit Complete Detection Kits 34081 SuperSignal West Pico Complete Kit Mouse IgG Detection Kit 34084 SuperSignal West Pico Complete Kit Rabbit IgG Detection Kit 34086 SuperSignal West Pico Complete Kit Biotinylated Protein Detection Kit For a list of kit components visit our website and search on the product For more information or to download product instructions visit www thermo com pierce Thermo Scientific SuperSignal West Dura Extended Duration Substrate Specially formulated for use with CCD cameras SuperSignal West Dura Extended Duration Substrate meets the needs of researchers using cooled charge coupled device CCD technology Cooled CCD cameras which offer the advantages of Instant image manipulation higher sensitivity greater resolution and a larger dynamic range than film eliminate the need for film processing equipment and a darkroom However this technology requires a substrate that produces an intense signal that is strong enough and of long enough duration to be captured by the camer
84. hang B et al 2003 Mol Cell Biol 23 5716 5725 Kaufmann S H et al 1987 Anal Biochem 161 89 95 Kaufmann S H and Kellner U 1998 Erasure of Western blots after autoradiographic or chemiluminescent detection In Immunochemical Protocols Ed Pound J D Humana Press Totowa NJ 223 235 Lanying Wen L et al 2003 Genetics 165 771 779 Schrager J A et al 2002 J Biol Chem 277 6137 6142 Skurk C et al 2004 J Biol Chem 279 1513 1525 Ordering Information Product Description Pkg Size 21059 Restore Western Blot Stripping Buffer 500 ml Sufficient for stripping 25 8 cm x 10 cm blots 21062 Restore Western Blot Stripping Buffer 30 ml Sufficient for stripping one 8 cm x 10 cm blot 21063 Restore Western Blot Stripping Buffer 5L Sufficient reagent to strip 500 8 cm x 10 cm blots Substrate The first blot A used the primary antibody diluted to 1 1 000 0 5 ug ml of Rat Anti Mouse IL 2 BD PharMingen San Jose CA and the horseradish peroxidase HRP labeled Goat Anti Rat secondary antibody Product 31470 diluted 1 5 000 The same blot was stripped with Restore Western Blot Stripping Buffer B for 5 minutes at room temperature and re probed C with the primary antibody at 1 5 000 and the HRP secondary conjugate at 1 20 000 SuperBlock Blocking Buffer was used for blocking Antibody 1 Stripped Antibody 2 Anti JAK 1 No Ab Clean Anti Bak Figure 3 Re probing with differ
85. he most out of your primary antibody three to 100 fold less primary antibody average Primary Antibody Reduction Factor PAR is 28 2 fold e Inexpensive costs approximately US 5 to treat an 8 x 10 cm blot e Conserves antibody regardless of detection system works with colorimetric chemiluminescent HRP and AP systems e Simple and ready to use fast 10 minute protocol Our Antibody Extender NC Promise Ordering Information Proper use of Pierce Antibody Extender NC will retain post transfer detection of your target protein on nitrocellulose Product Description Pkg Size membrane when using at least three times less primary 32110 itil anad Extender 500 ml antibody than you are currently using If you do not experience Sufficient reanentior up to aonitroceliilose a minimum of three fold reduction in primary antibody membranes 1 600 cm requirement with an equivalent or better performance on 32105 Pierce Antibody Extender 50 ml nitrocellulose membrane we will refund the cost of the reagent Solution NC Trial Pack Sufficient reagent to treat two nitrocellulose membranes 160 cr A simple 10 minute post transfer treatment of the target protein on nitrocellulose can reduce the amount of primary antibody used by three 10 25 and even 100 fold while maintaining equivalent signal compared to an untreated control How much will you save Primary Antibody Cost US 230 Primary Antibody Volume 200 ug Minimum Saving
86. hermo Scientific Pierce Reversible Protein Stain and Ponceau S Stain A comparison of GST lysate staining on nitrocellulose Increasing amounts of GST lysate protein were applied onto two 4 20 Tris glycine SDS polyacrylamide gels and electroblotted Blot A B Ponceau Stain Was treated with Pierce Reversible Stain for 30 seconds and destained according to the protocol Blot B was stained with 0 1 Ponceau S stain for 5 minutes and destained The blot stained with Pierce Reversible Stain demonstrates superior visual detec tion of bands GST lysate loading volumes Lane 1 3 Lane 1 5 pl Lane 2 10 ul Lane 3 15 ul and Lane 4 Pierce Prestained Protein MW Marker Product 26681 10 pl 1 2 3 4 12 3 4 A Thermo Scientific Pierce Reversible Stain 1 2 34 56 7 8 9 10 A Thermo Scientific Pierce B Ponceau S Stain Reversible Stain Figure 3 Comparison of Thermo Scientific Pierce Reversible Protein Stain with Ponceau S stain on PVDF membrane Pierce Unstained Protein MW Markers Product 26671 were serially diluted and applied to two 4 20 Tris glycine SDS polyacrylamide gels Lanes 1 9 Both gels were electroblotted to PVDF membrane Blot A was stained with Pierce Reversible Stain for 1 minute and destained according to the protocol Blot B was stained with 0 1 Ponceau S stain in 5 acetic acid for 5 minutes and destained according to the published protocol Lane 10 Pierce Prestained MW Marker Product 26681 1 2 3 4
87. hin polyacrylamide gels Bioluminescence and Chemiluminescence World Scientific Publishing Co pp 413 416 Roberts K P et al 2002 Biol Reprod 67 525 533 De loannes P et al 2004 Hemocyanin of the molluscan Concholepas concholepas exhibits an unusual heterodecameric array of subunits J Biol Chem 279 26134 26142 Roberts K P et al 2002 A comparative analysis of expression and processing of the rat epididymal fluid and sperm bound forms of Proteins D and E Biol Reprod 67 525 Wong W K P et al 2005 Bone morphogenetic protein receptor Type II C terminus interacts with c Src Implication for a role in pulmonary arterial hypertension Am J Respir Cell Mol Biol 33 438 446 To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor STEP STEP SDS PAGE Electro Tra r There are several methods for capturing data generated from chemiluminescent Western blots including X ray film cooled CCD cameras and phosphorimagers that detect chemiluminescence Cooled CCD cameras which offer the advantages of instant image manipulation greater resolution and a larger dynamic range than film also eliminate the need for a darkroom and film processing equipment Although electronic data capture with digital cameras and imagers is growing in popularity as the technologies improve and equipment prices decline most of the data obtained from Western blottin
88. ial handling Alternative labels are biotin fluorophores and enzymes The use of fluoro phores requires fewer steps and special equipment to view the fluorescence Also a photograph must be taken for a permanent record of the results Enzymatic labels are used most commonly and consistently produce excellent results Alkaline phosphatase AP and horseradish peroxidase HRP or POD are the two enzymes that are used extensively An array of chromogenic fluorogenic and chemiluminescent substrates is available for use with either enzyme For a detailed comparison of these two enzymes see Table 2 AP a 140 kDa protein that is generally isolated from calf intestine catalyzes the hydrolysis of phosphate groups from a substrate molecule resulting in a colored or fluorescent product or the release of light as a byproduct AP has optimal enzymatic activity at a basic pH pH 8 10 and can be inhibited by cyanides arsenate inorganic phosphate and divalent cation chelators such as EDTA STEP Formul KEEN ni h N Blockitt m ng N JD Wash Buff u C As a label for Western blotting AP offers a distinct advantage over other enzymes Because Its reaction rate remains linear detection sensitivity can be improved by simply allowing a reaction to proceed for longer HRP is a 40 kDa protein that catalyzes the oxidation of substrates by hydrogen peroxide resulting in a colored or fluorescent product or the release of light as a
89. ient reagent to strip one to two 8 cm x 10 cm blots 46430 Restore PLUS Western Blot 500 ml Stripping Buffer Sufficient reagent to strip 25 8 cm x 10 cm blots 46431 Restore PLUS Western Blot 5L Stripping Buffer Sufficient reagent to strip 500 8 cm x 10 cm blots Complementary Products 32106 Pierce ECL Substrate 500 ml 34080 SuperSignal West Pico Chemiluminescent 500 ml Includes Luminol Enhancer 250 ml Stable Peroxide Buffer 250 ml 34075 SuperSignal West Dura 100 ml Chemiluminescent Substrate Includes Luminol Enhancer Solution 50 ml Stable Peroxide Buffer 50 ml HRP Conjugated Goat Anti Rabbit HRP 1 ml HRP Conjugated Goat Anti Mouse HRP 1 ml 34095 SuperSignal West Femto 100 ml 1 Chemiluminescent Substrate Includes Luminol Enhancer Solution 50 ml Stable Peroxide Solution 50 ml HRP Conjugated Goat Anti Rabbit 1 ml HRP Conjugated Goat Anti Mouse 1 ml For more information or to download product instructions visit www thermo com pierce Enzyme Substrates Thermo Scientific Pierce Background Eliminator Another method by which the signal to noise S N ratio can be improved is to erase the background on exposed film leaving just the signal with little to no interference Pierce Background Eliminator does just that without altering the integrity of the data The Pierce Solution works on overexposed film lightening the entire film evenly This is done directly in the lab while viewing the film No darkroom is r
90. in Elmer products where applicable For more information or to download product instructions visit www thermo com pierce STEP Detection Reagents Thermo Scientific SuperSignal West Pico Substrate Stripping Buffer funooysajqnosy GE Healthcare Amersham ECL System 50 25 12 5 6 3 3 1 1 6 0 8 0 4 0 20 1 05 03 013 006 003 ng 50 25 1256 3 3 1 1 6 0 8 04 0 2 0 1 05 03 013 006 003 ng 1 minute Thermo Scientific SuperSignal West Pico Substrate 1 minute GE Healthcare Amersham ECL System 50 25 1256 3 3 1 16 08 04 0 2 0 1 05 03 013 006 003 ng 50 25 1256 3 3 1 16 08 04 0 2 0 1 05 03 013 006 003 ng 5 minutes 5 minutes Figure 4 Thermo Scientific SuperSignal West Pico is more sensitive than GE Healthcare Amersham ECL Substrate Recombinant mouse IL 2 was serially diluted 50 0 003 ng and electrophoresis was performed The gels were transferred to nitrocellulose membranes blocked and incubated with a 1 ug ml dilution of Rat Anti Mouse IL 2 After washing the membranes were incubated with 20 ng ml dilutions of HRP conjugated Goat Anti Rat antibody The membranes were washed again and then incubated with substrate that was prepared according to the manufacturers instructions Blots were exposed to film for one and five minute exposures Table 3 A conversion protocol for using Thermo Scientific SuperSignal West Pico Substrate Step by step GE Healthcare Amersham Conversion Protoc
91. inescent Substrate 34080 Supersignal West Pico 1 40 to 1 1 000 Chemiluminescent Substrate 32209 Pierce ECL Western 1 40 to 1 400 Blotting Substrate 34150 Lumi Phos WB Substrate 1 50 to 1 500 Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 _ L lt p53 l ij Wash Elute T Wash Elute Elute Control Control Immunoprecipitation Figure 3 Immunoprecipitation IP and Western blot experiments demon strate specificity of the Thermo Scientific Clean Blot IP Detection Reagent HRP Lane 1 A431 total cell extract expressing p53 positive control Lanes 2 and 3 No lysate negative control of IP wash Lane 2 and elution Lane 3 fractions Lanes 4 6 Complete IP experiment of wash Lane 4 and elution Lanes 5 and 6 fractions Lanes 1 5 were probed with Clean Blot IP Detection Reagent HRP and Lane 6 was detected with GAM HRP For more information or to download product instructions visit www thermo com pierce Enzyme Substrates Heavy Chain 55 kDa Cdk1 34 kDa Light Chain 22 kDa Figure 4 Easily distinguish your target protein on a Western blot with Thermo Scientific Clean Blot Detection Reagent HRP Mouse liver extract 50 ug total protein was separated on a Bio Rad Criterion Gel transferred to PVDF membrane and blocked with 5 milk in TBST The membrane was probed with mouse monoclonal anti Cdk1 LabVision 0 2 ug ml and goat anti mouse HRP 0 16 ug ml or Clean Blot Detection Reagent H
92. ing 50 gel lanes 22859 DyLight Infrared Protein 250 ul Molecular Weight Markers Sufficient material for loading 50 gel lanes t Patent pending on Dual labeled Fluorescent Molecular Weight Marker Technology STEP Formul Wash Buff Thermo Scientific Pierce Chemiluminescent Molecular Weight Markers New protein MW standard looks and acts like a typical pre stained marker for SDS PAGE and can also light up after transfer or in gel The Pierce Chemiluminescent Marker consists of seven proteins Spanning the molecular weight range from 18K to 220K Each marker component is covalently linked to a blue dye and chemically modified to impart peroxidase capability Unlike any other chemiluminescent detection compatible marker for Western blot applications Pierce Chemiluminescent Marker does not need an HRP antibody conjugate to yield a chemiluminescent signal A Colorimetric and B Chemiluminescent MW of Colorimetric and Component Detection ona Chemiluminescent Chemiluminescent Proteins Western Blot Markers In Gel Detection Myosin Heavy Chain Phosphorylase B BSA Ovalbumin Carbonic Anhydrase Trypsin Inhibitor Lysozyme Figure 3 On membrane and in gel detection using the Thermo Scientific Pierce Chemiluminescent Molecular Weight Markers These are representative MW values The covalently bound dye and enzyme alter the apparent MW of the component proteins relative to their unstained counterparts Lot speci
93. ing ample opportunity for mistakes to occur By stripping the membrane the blot can be reused After any stripping procedure the blot should be tested to ensure that all of the detection reagents were removed The membrane should be washed several times with blocking agent incubated with secondary antibody then reincubated with chemilumines cent substrate If the primary antibody was effectively removed by the stripping procedure no secondary antibody will bind to the membrane and no signal will be produced If bands are still visible on the blot the stripping conditions must be intensified Often a simple increase of the reaction time or temperature will complete the stripping process However it is sometimes necessary to alter the composition of the stripping buffer or change methods entirely To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor Electro Transfer Protocol for Stripping an Immunoblot Note 1 Optimization of both incubation time and temperature is essential for best results Note 2 If the blot cannot be stripped immediately after chemilu minescent detection store the blot in PBS at 4 C until ready to perform the stripping procedure 1 Place the blot to be stripped in Restore Western Blot Stripping Buffer and incubate for 5 15 minutes at RT Use a sufficient volume of buffer to ensure that the blot is completely wetted i e approxi m
94. ing without running another gel e After immunodetection the gel can be used for total protein staining there s no need to run two gels e The blocking step is omitted because the antibodies bind only specific antigens in the gel For more information or to download product instructions visit www thermo com pierce In Gel Detection Membrane Detection 1 2 3 4 5 6 7 1 2 3 4 5 6 7 High sensitivity using Thermo Scientific Pierce In Gel Detection Pure GFP 6xHis tagged and yeast GFP extract were separated by SDS PAGE One gel was transferred to nitrocellulose membrane After transfer the membrane was blocked overnight in 1 BSA The other gel was pre treated with 50 isopropanol Antigens were detected using a 1 1 000 dilution of polyclonal Anti Living Color Peptide Antibody Rabbit Clontech and the Pierce In Gel Chemiluminescent Detection Kit Rabbit Product 33500 Signal was detected using Pierce In Gel Detection Substrate The lanes on the gel and in the membrane are as follows Lanes 1 2 and 3 correspond to 10 5 and 1 ng pure GFP 6xHis tagged respectively Lanes 4 and 5 correspond to E coli bacterial GFP lysate diluted 1 100 and 1 1 000 respectively Lanes 6 and 7 correspond to yeast GFP Lysate diluted 1 10 and 1 100 respectively Note The Pierce In Gel Chemiluminescent Detection Kit has been tested successfully with Novex FMC BioWhittaker and Bio Rad Criterion brand gels e The Pierce In Gel Chemiluminescent
95. ited States contact your local branch office or distributor STEP STEP SDS PAGE Electro Transfer Ordering Information Product Description Pkg Size 34078 SuperSignal West Pico 1L Chemiluminescent Substrate Sufficient materials for 10 000 cm membrane Includes Luminol Enhancer 500 ml Stable Peroxide Buffer 500 ml 34087 SuperSignal West Pico 200 ml Chemiluminescent Substrate Sufficient materials for 2 000 cm membrane Includes Luminol Enhancer 100 ml Stable Peroxide Buffer 100 ml 34080 SuperSignal West Pico 500 ml Chemiluminescent Substrate Sufficient materials for 5 000 cm membrane Includes Luminol Enhancer 250 ml Stable Peroxide Buffer 250 ml 34077 SuperSignal West Pico 100 ml Chemiluminescent Substrate Sufficient materials for 1 000 cm membrane Includes Luminol Enhancer 2x 25 ml Stable Peroxide Buffer 2 x 25 ml 34079 SuperSignal West Pico 50 ml Chemiluminescent Substrate Trial Kit Sufficient materials for 500 cm membrane Includes Luminol Enhancer 25 ml Stable Peroxide Buffer 25 ml STEP STEP Formul Wash Buff Thermo Scientific SuperSignal Western Blotting Kits For convenience and ease of use nothing beats a complete Western blotting kit The Standard Detection Kits provide e HRP conjugated Anti Rabbit IgG Anti Mouse IgG or NeutrAvidin Biotin Binding Protein e SuperSignal West Pico Substrate The Complete Detection Kits provide e HRP conju
96. l 1 Direct in gel fluorescent detection Marker proteins 10 ul were separated in 4 20 Tris glycine gels and detected with the LI COR Odyssey Infrared Imaging System using intensity level 5 with the A 680 720 nm excitation emission setting B 780 820 nm excitation emission setting and C combined image Panel 2 Fluorescent detection on membranes Proteins were separated in 4 20 Precise Protein Gels and transferred to low fluorescence PVDF mem brane The membrane was blocked overnight in SEA BLOCK Blocking Buffer and imaged with the LI COR Odyssey System Panel 3 Colorimetric in gel detection Marker proteins 10 ul were separated in 4 20 Tris glycine gels and stained with A Imperial Protein Stain and B the Pierce Silver Stain Kit Il Panel 4 Fluorescent Western blot detection Marker proteins 5 ul were sepa rated in 4 20 Tris glycine gels and transferred to A nitrocellulose or B PVDF membrane Blots were imaged with the Typhoon 9410 at 500V PMT using the A Cy3 Fluor and B Cy5 Fluor laser settings Note Proteins in the marker mix produce uniform fluorescent intensities in SDS PAGE applications however variations in protein transfer efficiency affect intensity For example high MW proteins such as myosin 200K typically transfer less efficiently than low MW proteins Ordering Information Product Description Pkg Size 26665 DyLight Fluorescent Protein 250 ul Molecular Weight Markers Sufficient material for load
97. l 5 15 minutes Some antigen antibody systems require an increase in temperature and or longer incubation periods After determining that the membrane Is free of immunodetection reagents a second immunoprobing can begin Note 1 The Western blot can be stripped and reprobed several times but it may require longer exposure times or a more sensitive chemiluminescent substrate Subsequent reprobings may result in a decrease in signal if the antigen is labile in Restore Western Blot Stripping Buffer Analysis of the individual system is required Note 2 Reblocking of the membrane is not critical but it may be required in some applications STEP STEP Formul Blockit Wash Buff Perform SuperSignal Immunoassay 1 West Substrate Strip Blot with Restore Western Blot Stripping Buffer c N B Removal of A Removal of Primary Antibody HRP Conjugate and HRP Conjugate Supersignal Supersignal West Substrate West Substrate i HRP Perform Immunoassay 2 C gt mae SuperSignal West Substrate Figure 1 Thermo Scientific Restore Western Blot Stripping Buffer protocol For more information or to download product instructions visit www thermo com pierce Enzyme Substrates Thermo Scientific Restore Western Blot Stripping Buffer Strip time off your research with our stripping buffer Tired of re running electrophoresis gels and waiting to see your results Although optimizing assa
98. l and lowering the background by optimizing antibody concentration This process is made much easier by stripping and reprobing the membrane instead of starting from the beginning Stripping and reprobing a membrane One of the major advantages offered by chemiluminescent detec tion is the ability to strip reagents from a blot and then reprobe the same blot This is possible because the product detected is light rather than a colored precipitate on the membrane A blot may be stripped and reprobed several times to visualize other proteins or to optimize detection of a protein i e antibody concentrations without the need for multiple gels and transfers The key to this process is to use conditions that cause the release of antibody from the antigen without removing a significant amount of antigen from the membrane Various protocols have been proposed to accomplish this task and they generally include some combina tion of detergent reducing agent heat and or low pH During the stripping procedure some amount of antigen is inevitably removed from the membrane It is important to minimize this effect by stripping the antibody under gentle conditions Because each antibody antigen pair has unique characteristics there is no guaranteed method to remove every antibody while preserving the antigen Restore Western Blot Stripping Buffer Product 21059 and Restore PLUS Western Blot Stripping Buffer Product 46430 were designed to achieve maxi
99. l branch office or distributor SDS PAGE Electro Transfer Thermo Scientific Pierce O GlcNAc Western Blot Detection Kit High specificity monoclonal against 0 GIc NAc The Thermo Scientific Pierce O GlcNAc Western Blot Detection Kit contains the most highly specific mouse monoclonal antibody available for the detection of the O GlcNAc post translational modification Reaction of the monoclonal antibody in this Western blotting kit is confined to the B 0O linked serine or threonine GlcNAc modification There is no cross reactivity with the o O GIcNAc linkage the o B 0 GalNAc modification or the other N linked oligosaccharides Figure 9 Speed and sensitivity of chemiluminescent detection Chemiluminescent detection with SuperSignal West Dura Extended Duration Substrate allows visualization of 0 GIc NAc modified proteins in less than one minute after exposure of the blot to X ray film In addition to speed this kit is sensitive to the low picomole range Performance validated on Jurkat cell lysates This Western blot kit also includes the popular Thermo Scientific M PER Mammalian Cell Lysis Reagent an HRP labeled anti IlgM antibody conjugate blocking buffer and wash buffer components all validated to perform as specified Highlights e Kit includes MAb CTD 110 6 the most specific monoclonal antibody for the detection of B 0 linked N acetylglucosamine O GlcNAc e Detection of the target modification confined to only B
100. lectro Transfer Thermo Scientific Active GTPase Pull Down and Detection Kits Arf6 Cdc42 Rac1 RalA Rap1 Ras and Rho Monomeric p21 GTP binding proteins small GTPases serve as molecular switches in regulating a wide range of essential bio chemical pathways in eukaryotic cells Small GTPases are integral parts of cell physiology and are involved in several disease states such as cancer and metabolic disorders Like other G proteins small GTPases cycle between an inactive GDP bound state and an active GTP bound state The respective binding domain of the downstream effector for each small GTPase is expressed as a GSIT fusion protein which when immobilized on a resin is used to pull down the active GTP bound GTPase Figure 12 The pulled down active GTPase is then detected via Western blot using a specific antibody Each pull down kit contains all the necessary components for 30 pull down assays from 500 ug of cell lysate and a primary antibody for performing a Western blot Incubate for 1 hour at 4 C Active or GTP bound small GTPases Inactive or GDP bound small GTPases Non relevant proteins in the lysate 0000 Packed Resin T Fusion proteins Figure 12 Thermo Scientific Active GTPase Pull Down and Detection Kit protocol summary Complete kits for pulling down and detecting small GTPases Arf1 STEP STEP Formul Wash Buff Blocki Highlights e Convenient no need to ex
101. lorimetric Substrates conjugated antibodies blocking buffers and standard buffers e Pierce Chloronaphthol Product 34012 STEP 5 e TMB Blotting Product 34018 Primary and Secondary Detection Reagents e NBT BCIP Product 34042 Incubate the membrane with antibody e Metal Enhanced DAB Product 34065 3 For a complete list visit the secondary antibody selection guide at www thermo com pierce For a complete listing of primary antibodies request a copy of the Thermo Scientific Pierce Antibody Handbook featuring over 35 000 antibodies in 42 research areas or visit www thermo com abr Film La Expose the membrane to X ray film e CL XPosure Film 5 x 7 sheets Product s 34090 and 34092 For direct detection methods 8 x 10 sheets Product s 34091 and 34093 18 x 24 cm sheets we offer Product 34089 e Monoclonal Antibodies e Pierce Background Eliminator Kit Product 21065 e Fluorescent Probes and Labeling Kits e Enzyme Labeling Kits For indirect detection methods we offer e Biotinylation Kits Stripping Buffer e Protein A Protein G and Protein L labeled with fluorescein Reprobe the blot if necessary rhodamine HRP AP or biotin e Avidin Streptavidin and NeutrAvidin Biotin Binding Protein e Restore Western Blot Stripping Buffer labeled with fluorescein rhodamine HRP or AP Product 21059 and 21063 e Secondary antibodies labeled with fluorescein rhodamine e Restore PLUS Western Blottin
102. lot is exposed to film e Make sure membranes are wetted thoroughly and according to the manufacturer s instructions e Use new membranes e Ensure the membrane is adequately covered with liquid at all times to prevent it from drying e Use agitation during all incubations e Handle membranes carefully damage to the membrane can cause nonspecific binding s Do not handle membrane with bare hands Always wear clean gloves or use forceps e Prepare new buffers For more information or to download product instructions visit www thermo com pierce Enzyme Substrates D S o 2 g g E C 8 a E ne na ay eh S Sea a ae m P PT K a a lt a Fa a ran aka La hR m 2 KE aa on IC n aGuRY LOR OR EEO a Gee J J ET J L Se bo A a Vw Z i ae l i wa Sex Possible Causes Precautions Solutions Antibody concentrations are too high s The primary and or secondary antibody can cause high background if the concentrations used are too high e Decrease antibody concentrations Aggregate formation in the HRP e Filter the conjugate through a 0 2 um filter conjugate can cause speckling e Use a new high quality conjugate Incompatable blocking buffer was used lt Compare different blocking buffers Insufficient blocking of e Optimize blocking buffer The best blocking buffer is system dependent nonspecific sites e Increase concentration of protein in the blocking buff
103. ls in Protein Science pp 10 10 1 10 10 11 John Wiley and Sons Inc New York Gershoni J 1988 Protein blotting A manual Meth Biochem Anal 33 1 58 Gershoni J M and Palade G E 1983 Protein blotting Principles and applications Anal Biochem 131 1 15 Gershoni J M and Palade G E 1982 Electrophoretic transfer of proteins from sodium dodecyl sulfate polyacrylamide gels to a positively charged membrane filter Anal Biochem 124 396 405 Harlow E and Lane D 1988 Antibodies A Laboratory Manual Cold Spring Harbor Laboratory Cold Spring Harbor New York Product 15050 Malik V S and Lillehoj E P 1994 Antibody Techniques Academic Press Inc San Diego CA Ramlau J 1987 Use of secondary antibodies for visualization of bound primary reagents in blotting procedures Electrophoresis 8 398 402 Spinola S M and Cannon J G 1985 Different blocking agents cause variation in the immunologic detection of proteins trans ferred to nitrocellulose membranes J mmunol Meth 81 161 165 Towbin H et al 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets procedure and some applications P Natl Acad Sci USA 76 4350 4354 Ursitti J A et al 1995 Electroblotting from Polyacrylamide Gels Current Protocols in Protein Science pp 10 7 1 10 7 14 John Wiley and Sons Inc New York Young P R 1989 An improved method for the det
104. lue purple PPT 1 1 250 100 pg AP 2 1 2 5K Pierce NBT Substrate 34035 Blue purple PPT 1 1 250 100 pg AP 2 1 2 5K Pierce NBT BCIP Substrate 34042 Black purple PPT 1 1 500 30 pg AP 2 1 2 5K Pierce NBT BCIP Suppressor Substrate 34070 Black Purple PPT 1 1 500 30 pg AP 2 1 2 5K Actual sensitivity is unique to each antibody antigen pair The approximate sensitivities listed are conservative amounts that should be easily detectable for most antigens 1 Primary 2 Secondary PPT precipitate HRP horseradish peroxidase AP alkaline phosphatase To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor STEP STEP Formul Electro Trangrer Wash Buff Thermo Scientific Pierce Fast Western Blot Kit consumables required by other popular rapid Western blotting ECL Substrate systems No vacuum pump is needed so there are never any clogged lines or membranes that can occur when using instrument based systems Because there is no apparatus there is no need to buy disposable blotting trays that limit the number of blots processed at one time The Pierce Fast Western Blot Kit ECL Substrate contains optimized reagents that shorten the time to perform a typical Western blot from 4 hours down to approximately 55 minutes The kit provides all the reagents necessary to complete a Western blot being Highlights probed with a mouse or rabbit p
105. luor 647 and Cy5 Dyes Near IR 680 692 712 140 000 Alexa Fluor 680 and Cy5 5 Dyes Near IR 750 752 778 220 000 Alexa Fluor 750 and Cy7 Dyes Near IR 800 777 790 270 000 IRDye 800 Dye Excitation and emission maxima in nanometers 4 nm T Molar extinction coefficient MT cm DyLight 405 DyLight 488 DyLight 549 350 400 450 500 550 600 650 700 750 800 850 Wavelength nm 350 400 450 500 550 600 650 700 750 800 850 Wavelength nm 350 400 450 500 550 600 650 700 750 800 850 Wavelength nm DyLight 594 DyLight 633 DyLight 649 A 350 400 450 500 550 600 650 700 750 800 850 Wavelength nm Excitation Emission Excitation Emission Excitation 350 400 450 500 550 600 650 700 750 800 850 Wavelength nm 350 400 450 500 550 600 650 700 750 800 850 Wavelength nm DyLight 680 DyLight 750 DyLight 800 S S 5 S S S A A L A 350 400 450 500 550 600 650 700 750 800 850 Wavelength nm 350 400 450 500 550 600 650 700 750 800 850 Wavelength nm 350 400 450 500 550 600 650 700 750 800 850 Wavelength nm Excitation and emission spectra for Thermo Scientific DyLight Dyes Emission To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor 27 Electro Transfer Thermo Scientific Clean Blot IP Detection Reagents Clearly better Western blots Antibody bands often mask target proteins when performing Western blots on immunoprecipitated sam
106. membrane The blocking buffer should improve the sensitivity of the assay by reducing background interference Individual blocking buffers are not compatible with every system For this reason a variety of blockers in both Tris buffered saline TBS and phosphate buffered saline PBS are available The proper choice of blocker for a given blot depends on the antigen and on the type of enzyme conjugate to be used For example with applications using an alkaline phos phatase conjugate a blocking buffer in TBS should be selected because PBS interferes with alkaline phosphatase The ideal blocking buffer will bind to all potential sites of nonspecific inter action eliminating background without altering or obscuring the epitope for antibody binding For true optimization of the blocking step for a particular immuno assay empirical testing is essential Many factors can influence nonspecific binding including various protein protein interactions unique to a given set of immunoassay reagents The most impor tant parameter when selecting a blocker Is the signal to noise ratio which is measured as the signal obtained with a sample containing the target analyte as compared to that obtained with a sample without the target analyte Using inadequate amounts of blocker will result in excessive background noise and a reduced signal to noise ratio Using excessive concentrations of blocker may mask antibody antigen interactions or inhibit the marker e
107. mum removal of antibodies while preserving the integrity of the antigen They are unique among stripping buffers because they are odor free and can often strip a membrane in as little as 15 minutes Figure 1 next page Stripping and reprobing a Western blot instead of preparing an entirely new blot may be preferable because it e Conserves sample When the protein mixture Is rare or valuable reprobing con serves the sample and allows the membrane to be analyzed with the same or different antibodies Saves time It is time consuming to run an SDS polyacrylamide gel and then transfer the proteins to a membrane By using the same blot for several different detections you save time e Makes it easy to optimize The light emission intensity of SuperSignal West Pico Substrate and the increased sensitivity of SuperSignal West Dura and SuperSignal West Femto Substrates often require antibody concentration optimization to achieve the highest quality blot Optimization is achieved easily by stripping the membrane and reprobing with a different antibody concentration Saves money By reusing the same blot you save money on the costs of membrane buffers and protein sample Makes it easy to confirm atypical results When immunoblot results are not as expected reprobing allows the use of the same protein sample without going back to gel electrophoresis e Makes it easy to correct mistakes Immunoblotting requires many steps provid
108. n durable cards enabling them to be used easily at the bench This helpful guide along with high quality Thermo Scientific Pierce Products will help you purify immobilize label and store antibodies and perform common procedures such as immuno precipitation Western blotting and ELISA Ordering Information Product Description Pkg Size 15051 Using Antibodies 1 book A Laboratory Manual Ed Harlow and David Lane Published by Cold Spring Harbor Laboratory Press 1999 495 pages wire spiral bound hardcover with nine separate portable protocols Sorry books are nonreturnable 24 STEP STEP Blockin d i Formul Wash Buff 9 Antibody Stabilizers and Storage Solutions Polyclonal secondary antibodies are typically stable when stored frozen as concentrated stock solutions in simple phosphate or Tris buffers containing sodium azide or other antimicrobial agents Most uses of secondary antibodies require more than 1 000 fold dilution using only a few microliters of stock and the daily need for Western blotting or ELISA experiments inevitably leads to repeated freeze thaw cycles that are damaging to the antibody and conjugated enzyme Freeze thaw cycles can be avoided by storing a concentrated antibody stock as a mixture containing 20 50 of an anti freeze compound such as glycerol or ethylene glycol Glycerol is most frequently used for this purpose but commonly contains impu rities that can adver
109. n that was originally derived from the bacteria Peptostreptococcus magnus but now Is produced recombinantly in E coli e Has the unique ability to bind through kappa light chain interactions including kappa I III and IV in human and kappa in mouse without interfering with an antibody S antigen binding site Ordering Information Product Description Pkg Size 21189 Pierce Purified Recombinant 1 mg Protein Lyophilized 32420 Protein L Peroxidase Conjugated 0 5 mg 29997 Biotinylated Protein L 0 5 mg For more information or to download product instructions visit www thermo com pierce Enzyme Substrates Thermo Scientific Avidin Biotin Products The interaction between biotin a vitamin and avidin hen egg white protein has been exploited to produce a variety of applications The noncovalent high affinity of biotin for avidin K 10 M has allowed us to create a line of products that can help you develop nonradioactive assay systems With four biotin binding sites per avidin molecule this system allows more signal to be concentrated at the detection site A similar assay scenario can be developed for DNA or RNA hybridization assays when a probe is biotinylated instead of an antibody Below are just a few of the applications exploiting the avidin biotin interaction even beyond assay development e ELISA e Immunohistochemical staining e Western blotting e DNA hybridization assays e Immunoprecipitation
110. nate Buffer Pack Product 28388 For more information or to download product instructions visit www thermo com pierce STEP 4B Formulate Wash Buffers Add detergent to blocking wash buffers to reduce nonspecific binding Enzyme Substrates Add the detection reagent of erSignal ibstrate T NS Skip this step if you use StartingBlock T20 d Blocking Buffer in PBS Product 37539 or TBS Product 37543 or SuperBlock Chemiluminescent Substrates T20 Blocking Buffer in PBS Product e Pierce ECL Substrate 37516 or TBS Product 37536 These Product s 32106 32209 and 32109 buffers already contain Tween 20 Detergent at optimized concentrations e Pierce Fast Western Blot Kit ECL Substrate containing e SuperSignal West Pico Chemiluminescent Substrate e Tween 20 Product 28320 Product s 34077 and 34080 also available in an and Tween 80 Product 28328 economical 1 L package Product 34078 e Triton X 100 Product 28314 and Triton X 114 e SuperSignal West Femto Maximum Sensitivity Product 28332 Substrate Product s 34096 and 34095 e Nonidet P 40 Product 28324 e SuperSignal West Dura Extended Duration Substrate e Brij 35 Product 28316 and Brij 58 Product 28336 Product s 34076 and 34075 e Lumi Phos WB Substrate Product 34150 For convenience and economy we also offer complete Western blotting kits that include chemiluminescent substrates enzyme Co
111. nd related procedures Thermo Scientific SuperSignal Technology is protected by U S patent 6 432 662 Thermo Scientific Slide A Lyzer MINI Dialysis Unit Technology is protected by U S patent 6 039 871 Thermo Scientific Pierce Direct Detection of Biomolecules Technology is protected by U S patent 7 112 411 Thermo Scientific SwellGel Technology is protected by U S patent 6 709 743 U S patents pending on Thermo Scientific Pierce Western Blot Signal Enhancer Technology and DyLight Dual Labeled Protein Molecular Weight Marker Technology Bio Rad Molecular Imager Criterion PROTEAN and Ready Gel are trademarks of Bio Rad Laboratories Inc Chemilmager is a trademark of Alpha Innotech Corporation Kathon and Triton are trademarks of Rohm amp Haas Company Lumi Phos is a trademark of and is sourced from Lumigen Inc Alexa Fluor Surelock Novex iBlot and NuPAGE are trademarks of Invitrogen Corporation BioMax and X Omat are trademarks of Eastman Kodak Company Tween and Brij are trademarks of ICI Americas Inc Typhoon Storm Cy Cy3 Cy5 CyDye ECL Plus Hyperfilm and Hybond are trademarks of GE Healthcare Cascade Blue and Texas Red are trademarks of Molecular Probes Inc Gels is a trademark of Gradipore Ltd Odyssey and IRDye are trademarks of LI COR Biosciences Western Lightning is a trademark of PerkinElmer Inc SNA
112. ndary Antibody 1 5 000 1 50 000 High background and or unwanted bands are often caused by using too much enzyme 5 Bands glow visibly directly on the membrane this should never occur and will certainly over expose sensitive X ray film To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor STEP STEP SDS PAGE Electro Tra r Optimize Antibody Concentration Because every new Western blot is unique there is no perfect antibody concentration for every blot Therefore every new Western blot needs to be optimized to determine the antibody concentration that is most appropriate for a particular combination of membranes proteins and antibodies Optimization is even more crucial when key components of a system are changed such as switching from a colorimetric substrate like chloronaphthol CN to more sensitive chemiluminescent substrates such as SuperSignal West Products Antibodies must be used at the optimal concen trations with chemiluminescent substrates to achieve low back ground and high band resolution Figures 2 3 The first step of optimizing the blotting conditions usually involves optimizing the antibody concentrations or dilutions through the use of a dot blot protocol The next step is typically the optimization of the blocking buffer by testing cross reactivity of several different buffers with the blotting system s key components see page 13
113. ne Western blotting also called immuno blotting because an antibody is used to specifically detect its antigen was introduced by Towbin et al in 1979 and is now a routine technique for protein analysis he specificity of the antibody antigen interaction enables a target protein to be identified in the midst of a complex protein mixture Western blotting can produce qualitative and semiquantitative data about that protein The first step in a Western blotting procedure is to separate the macromolecules using gel electrophoresis After electrophoresis the separated molecules are transferred or blotted onto a sec ond matrix generally a nitrocellulose or polyvinylidene difluoride PVDF membrane Next the membrane is blocked to prevent any nonspecific binding of antibodies to the surface of the membrane The transferred protein is complexed with an enzyme labeled antibody as a probe An appropriate substrate is then added to the enzyme and together they produce a detectable product such as a chromogenic precipitate on the membrane for colorimetric detection The most sensitive detection methods use a chemi luminescent substrate that when combined with the enzyme produces light as a byproduct The light output can be captured using film a CCD camera or a phosphoimager that is designed for chemiluminescent detection Whatever substrate is used the intensity of the signal should correlate with the abundance of the antigen on the blotting mem
114. nes were washed and incubated with substrates that were prepared according to the manufacturer s instructions Each membrane was exposed to X ray film for 5 minutes The SuperSignal West Dura Substrate membrane was exposed to the Chemilmager 4000 for 30 minutes 5B and the GE Healthcare ECL Plus Blot was exposed for 15 minutes 5D Reference Tokumaru H et al 2001 Cell 104 421 432 Ordering Information Product Description Pkg Size 34076 SuperSignal West Dura 200 ml Extended Duration Substrate Sufficient materials for 2 000 cm membrane Includes Luminol Enhancer 100 ml Stable Peroxide Buffer 100 ml 34075 SuperSignal West Dura 100 ml Extended Duration Substrate Sufficient materials for 1 000 cm membrane Includes Luminol Enhancer 50 ml Stable Peroxide Buffer 50 ml 37071 SuperSignal West Dura Extended 20 ml Duration Substrate Trial Kit Sufficient materials for 200 cm membrane Includes Luminol Enhancer 10 ml Stable Peroxide Buffer 10 ml To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor Electro Transfer Thermo Scientific SuperSignal West Femto Maximum Sensitivity Substrate True femtogram detection SuperSignal West Femto Maximum Sensitivity Substrate provides the ultimate sensitivity for Western blotting allowing you to see protein bands that were never before visualized Highlights e Sensitive reach low femtogram
115. nese and selenium Thermo Scientific SuperBlock StartingBlock and Protein free Blocking Buffer Ingredients A single protein or protein alternative PBS or TBS buffer and a preservative For more information or to download product instructions visit www thermo com pierce Enzyme Substrates The most appropriate blocking buffer for Western blotting use Blocking Buffers Application Chart is often system dependent Determining the proper blocking Immuno DNA RNA buffer can help to increase the system s signal to noise ratio Blocking Western Dot histo Hybridiza Occasionally when switching from one substrate to another the AUR Biter Se ome ions blocking buffer that you are using will lead to diminished signal or eo Bese oe Zo a increased background Empirically testing various blocking Blocking Buffer buffers with your system can help achieve the best possible 37542 StartingBlock VY v Z v results Avoid using milk as a blocking reagent for blots that rely on TBS the avidin biotin system because milk contains variable amounts Blocking Buffer of biotin Although SuperBlock Blocking Buffer Product 37515 37539 StartingBlock vY v vv often gives excellent results we recommend testing several block 120 PBS Br ee s s Blocking Buffer ing reagents for their suitability in a particular system There is no 37543 StartingBlock v A YV 4 blocking reagent that will be the optimal reagent for all systems T20 TBS l l i Bl
116. nsfer protein from gel to membrane in 7 10 minutes Sensitive same transfer efficiency as other semi dry transfer units or traditional wet transfer units Economical no consum ables needed Robust transfer up to 4 gels at one time Versatile use homemade or pre cast gels Optimized formulated for accelerated transfer Non hazardous no methanol required eliminating hazardous disposal Compatible use with all major SDS PAGE gels Tris HEPES Tris Glycine Bis Tris and Tris HCl Flexible use with any semi dry transfer unit use with nitrocellulose or PVDF membranes Easy to use simply dilute the 10X concentrated formula in ultrapure water 1 hour to overnight Cost per transfer Consumables Yes transfer stacks No Environmentally friendly Yes methanol not No requires dis required no disposal posal of cartridges of consumables Up to 4 gels per run Up to 2 gels per run No requires addi tion of methanol Throughput Up to 2 gels per run To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor STEP SDS PAGE STEP Electro Ira r NJ iBlot Transfer System Pierce Fast Transfer System ug 16 8 4 2 16 8 4 2 PRKDC 350 kDa EGFR 170 kDa PLK1 67 kDa CDC2 34 kDa Cyclophilin B 21 kDa Transfer time 7 10 minutes 7 minutes Transfer cost
117. nto the gel onto the gel Possible Causes Precautions Solutions Antibody concentrations are too high Reduce antibody concentrations especially the HRP conjugate Signal that decreases quickly and the appearance of white bands are indications that there is too much HRP in the system Possible Causes Precautions Solutions Incomplete transfer of proteins e Make sure there are no air bubbles between the gel and membrane during transfer from the gel e Wet membrane according to the manufacturer s instructions e Do not handle the membrane with bare hands Always wear clean gloves or use forceps e Use a new membrane e ncubate membranes separately to ensure that membrane strips are not covering one another during incubations To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor funooysajqnosy STEP STEP STEP STEP Formul Electro Trangrer Wash Buff Full Length Western Blotting Protocol Using 5 Remove the membrane blot and block the nonspecific sites with Ch lumi t Substrat a blocking buffer for 20 60 minutes at RT with shaking For best emiltuminescent substrates results block for 1 hour at RT Optimization of blocking buffer may be required to achieve best results Please see the Blocking 1 Make th tein solution of i i E i ake the protein solution of interest in a sample buffer and heat Buffer Optimization section page 13 it to b
118. nzyme again causing a reduction of the signal to noise ratio When developing any new immunoassay It is important to test several different blockers for the highest signal to noise ratio in the assay No single blocking agent is ideal for every occasion because each antibody antigen pair has unique characteristics Ifa blocking buffer that does not cross react with your system cannot be found Pierce In Gel Protein Detection is an alternate choice This method specifically detects proteins within the gel and requires no blocking see page 50 for more information We offer a complete line of blocking buffers for Western blotting including BLOTTO Casein BSA SEA BLOCK and the exclusive SuperBlock and StartingBlock Blocking Buffers Formulage Wash Buff aid STEP e S Which blocking buffer is most likely to cause a high background Nonfat Dry Milk Ingredients B lactoglobulin lactalglobulin antibodies serum albumin three or more different caseins enzymes hormones growth factors nutrient transporters disease resistance factors entire leukocytes other proteins lactose glucose galactose amino sugars sugar phosphates neutral and acid oligonucleotides nucleotide sugars monosaturated fatty acids polyunsaturated fatty acids saturated fatty acids A B 6 B 12 D E H biotin folate niacin pantothenic acid riboflavin thiamin calcium iron magnesium phosphorous potassium sodium zinc copper manga
119. ocking Buffer Various proteins were analyzed by Western blotting to determine 37515 Te S z J S the optimal blocking condition for nonspecific sites Figure 1 The Blocking Buffer resulting blots were analyzed for signal to noise and compared in PBS The results indicate that there is no single blocking reagent that is 37535 SuperBlock Y v Y v v optimal for all systems Blocking Buffer in TBS 37517 SuperBlock v YV Y Thermo Scientific Blocking Buffer SuperBlock Blotting in PBS Blocking Buffer Milk Casein BSA 37537 SuperBlock Y YV vY 1 50 1 10 1 2 1 50 1 10 1 2 1 50 1 10 1 2 1 50 1 10 1 2 Blocking Buffer Cyclin B1 Blotting in TBS 30 Second a0 20 ag 37516 SuperBlock vV v Y v v Exposure T20 PBS p53 x s 21 Blocking Buffer 30 Second a N 37536 SuperBlock Y v Y Y v Exposure k T20 TBS fos Blocking Buffer 30 Second af s 37527 SEABLOCK v v v Exposure __ ME SESS Blocking Buffer fos 37520 Blocker BSA VY 112 WV v v 5 Minute lt 0 Ba in TBS Exposure GE FEO S O 3 Blocker BSA V V V y v Figure 1 Blocking buffer optimization Recombinant human cyclin B1 EES wild type p53 and mouse fos baculovirus lysates were diluted in Lane Marker 37532 Blocker Y 2 o E Reducing Sample Buffer 1 50 1 10 or 1 2 and separated electrophoretically Casein in TBS on a 12 SDS polyacrylamide gel The proteins were transferred to 37528 Blocker yo v vv Y nitrocellulose membrane and cut into strips The membrane strips were Casein in PBS
120. oiling for 5 minutes The sample buffer should contain the following 6 0 03 M TriseHCl 5 SDS to denature the protein and to generate a constant anionic charge to mass ratio for the denatured protein chains Incubate the blot with the primary antibody with shaking for 1 hour For recommended antibody dilutions see the table below If desired blots can be incubated with primary antibody overnight at 2 C 8 C The necessary dilution will vary depending on the primary antibody used and the amount of antigen that was transferred Please see the Optimize Antibody Concentration section page 60 50 glycerol to give the sample a higher density than the running buffer allowing the sample to sink to the bottom of the well 3 i at Recommended Primary Antibody A low MW dye for dye front determination Thermo Scientific Substrate Dilutions from 1 mg ml stock e As needed a reducing agent such as 100 mM ane Pierce ECL Substrat 1 100 1 5 000 or 0 2 10 2 mercaptoethanol dithiothreitol or TCEP that will reduce dai ai pg m the disulfide bonds present in the protein sample SuperSignal 1 1 000 1 5 000 or 0 2 1 0 pg ml l l West Pico Substrate Adjust solution to pH 6 8 SuperSignal 1 5 000 1 100 000 or 0 01 0 2 ug ml 2 Add the protein solution in the sample buffer to an West Femto Substrate SDS polyacrylamide gel SuperSignal 1 1 000 1 50 000 or 0 02 1 0 ug ml West Dura Substrate 3 Separate the proteins electrophoretically b
121. ol ECL Substrate 1 Perform standard Use their Hybond electrophoresis and blotting Nitrocellulose Membrane 2 Block the nonspecific sites Add blocking reagent incubate and wash 3 Add diluted primary antibody Optimization Range incubate for 1 hour then wash 1 100 1 1 500 dilution 4 Add diluted secondary Optimization Range antibody HRP labeled 1 1 500 1 50 000 dilution incubate for 1 hour then wash 5 Prepare chemiluminescent Mix equal volumes of both solutions substrate 6 Incubate the substrate Incubate blot with Working Solution on the blot without agitation for precisely 1 minute It s recommended that you work quickly once GE s ECL Working Solution has been added to the membrane 7 Expose to film Immediately expose to film for 1 minute References Ju T et al 2002 J Biol Chem 277 178 186 Kagan A et al 2000 J Biol Chem 275 11241 11248 Messenger M M et al 2002 J Biol Chem 277 23054 23064 Thermo Scientific SuperSignal West Pico Substrate Use any nitrocellulose or PVDF membrane Add blocking reagent incubate and skip the wash Optimization Range 1 1 000 1 5 000 dilution Optimization Range 1 20 000 1 100 000 dilution Mix equal volumes of both solutions Incubate blot with Working Solution with agitation for 5 minutes The signal lasts for hours so take your time Expose to film for 1 minute To order call 800 874 3723 or 815 968 0747 Outside the Un
122. oles fluorescein mole avidin e Fluorescent labeled avidin e Ex Em 450 570 nm and 574 nm Applications e Immunoassay reagent when bound to biotinylated enzymes or when conjugated to enzymes e Blocking protein for biotin rich tissue sections use at 0 1 for inhibition of endogenous biotin e Use in immunohistochemistry where endogenous phosphatase is a problem e Western blotting e Use for immunohistochemistry where high levels of endogenous peroxidase is a problem e Western blotting e ELISA e Fluorescence activated cell sorting FACS e Histochemical staining e Fluorescence activated cell sorting FACS e Histochemical staining Pkg Size 10 mg 20 mg 2 mg 5 mg 100 units 5 mg 1 mg To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor STEP STEP SDS PAGE Electro Trangrer As with the other components in a Western blotting system there are many substrate choices available The appropriate sub strate choice depends on the enzyme label AP or HRP desired sensitivity and desired form of signal or method of detection Chromogenic substrates are widely used and offer perhaps the simplest and most cost effective method of detection When these substrates come in contact with the appropriate enzyme they are converted to insoluble colored products that precipitate onto the membrane and require no special equipment for processing or
123. on Reagents are the perfect substitute for traditional secondary antibody conjugates These unique conjugates recognize most primary antibodies independent of the host species Table 6 and can be used with IPs performed using Protein A or G agarose resins This versatility eliminates the need to buy separate detection kits based on primary antibody species Our conjugates are conveniently stored at 2 8 C and are compatible with any HRP or AP substrate including Thermo Scientific Pierce ECL SuperSignal Chemiluminescent and Lumi Phos WB Substrates Table 7 For added convenience the HRP conjugate is available in a kit that contains StartingBlock T20 Blocking Buffer and Pierce ECL Chemiluminescent Substrate STEP Blocki STEP Formul Wash Buff Table 6 Thermo Scientific Clean Blot IP Detection Reagents recognize the various polyclonal antibodies and the specific monoclonal antibodies listed To determine specific antibody compatibility perform a dot blot analysis Species Monoclonal Isotype s Bovine IgG Goat IgG Human IgG IgG IgG Mouse IgG 19G 196 Rat IgG Sheep IgG Table 7 Recommended dilution ranges for the Thermo Scientific Clean Blot IP Detection Reagents when using our chemiluminescent substrates Recommended Product Chemiluminescent Substrate Dilution Range 34095 Supersignal West Femto 1 200 to 1 4 000 Chemiluminescent Substrate 34075 Supersignal West Dura 1 200 to 1 2 000 Chemilum
124. one step probe incubation eliminates the lengthy two step primary secondary antibody sequential reaction protocol e Sensitive when used in combination with SuperSignal West Chemiluminescent Substrates this kit allows the detection of even low expression histidine tagged clones A B e More versatile than anti polyHis antibody based systems the HisProbe Kit detects polyhistidine fusion proteins that are undetectable using some monoclonal anti polyHis antibodies e Sufficient reagents for fifty 7 5 x 10 cm blots Figure 8 Specificity comparison of polyhistidine tagged PHT fusion protein detection methods Panel A using HisProbe HRP shows high specific binding and low background Panel B using anti polyHis failed to recognize two of the three fusion proteins References Adler J and Bibi E 2004 J Biol Chem 279 8957 8965 Kanaya E et al 2001 J Biol Chem 276 7383 7390 Kiick K L et al 2002 P Natl Acad Sci USA 99 19 24 Sylvester S R and Roy A 2002 Biol Reprod 67 895 899 Ordering Information Product Description Pkg Size 15165 HisProbe HRP 2 mg 15168 SuperSignal West Pico Kit HisProbe Kit Includes HisProbe HRP 2 mg SuperSignal West Pico 500 ml Chemiluminescent Substrate Blocker BSA in TBS 10X 1x 125 ml BupH Tris Buffered Saline Packs 10 x 500 ml Surfact Amps 20 10 Ampules 6 x 10 ml To order call 800 874 3723 or 815 968 0747 Outside the United States contact your loca
125. optimization of the amount of enzyme normally an HRP conjugated secondary antibody in the system The amount of enzyme is affected by a variety of factors the most important of which are the amount of primary and sec ondary antibody used Optimization of the antibody concentration Is discussed on pages 60 63 The most important aspect to remember when using a chemilumi nescent substrate is that too much enzyme is detrimental to signal development This is counter intuitive to many people especially to those accustomed to blotting with colorimetric systems in which increasing the amount of enzyme increases the amount of color generated In a colorimetric system the enzyme permanently con verts a non colored substrate into a precipitated colored byproduct but this is not what happens in chemiluminescent systems In chemiluminescent systems the enzyme HRP converts the substrate luminol into a product that temporarily emits light How much light is generated and how long the signal lasts depends on the ratio of the enzyme to the substrate The amount of substrate Is relatively constant but the amount of enzyme changes depending on how much someone adds If not enough enzyme is added then no signal is generated If too much enzyme is added the reaction between the enzyme and the substrate occurs so rapidly that there Is a flash of light that can last mere seconds Figure 1 The signal terminates before a picture can be taken Too m
126. ormation Product Description Pkg Size 22854 DyLight 549 649 Western Blotting Kit Kit Sufficient reagents for 10 Western blots Includes DyLight 549 Goat Anti Mouse IgG H L 50 ul DyLight 649 Goat Anti Rabbit IgG H L 50 ul Fluorescent Dual labeled 60 pl Protein MW Markers Wash Buffer 30X 200 ml Blocker BSA in PBS 10X 50 ml Low Fluorescence PVDF Transfer Membrane 10 each Complementary Product 22860 Low Fluorescence PVDF Transfer Membrane 0 2 um 7 cm x 8 4 cm 10 pkg Thermo Scientific DyLight 680 800 Near Infrared Western Blotting Kit Compatible with LI COR Odyssey and other fluorescent imaging systems Near infrared fluorescent detection of two different targets on a single Western blot is easy to perform with the Thermo Scientific DyLight 680 800 Western Blotting Kit The kit provides highly optimized reagents and a convenient format to save you the time and frustration of having to evaluate reagents for compatibility with fluorescent Western blotting Figure 11 Each kit contains sufficient reagents for 10 Western blots and includes Pierce Dual Labeled Protein Molecular Weight Markers and secondary antibodies conjugated to DyLight 680 and 800 Fluorescent Dyes Highlights e DyLight 680 excitation emission maxima 692 712 nm e DyLight 800 excitation emission maxima 777 790 nm e Optimized format provides low background and high signal intensity e Convenient saves time and money associated wi
127. ormulation in phosphate buffered saline pH 7 5 for use in Western blotting and ELISA applications 37542 StartingBlock TBS Blocking Buffer 1L A protein based blocker formulation in Tris buffered saline pH 7 5 for use in Western blotting and ELISA applications StartingBlock Blocking Buffers are also available with an optimized amount of Tween 20 Detergent to provide the lowest background Ordering Information Product Description Pkg Size 37539 StartingBlock T20 PBS Blocking Buffer 1L A protein based blocker formulation in phosphate buffered saline at pH 7 5 with 0 05 Tween 20 Detergent and Kathon Antimicrobial Agent 37543 StartingBlock T20 TBS Blocking Buffer 1L A protein based blocker formulation in Tris buffered saline at pH 7 5 with 0 05 Tween 20 Detergent and Kathon Antimicrobial Agent Thermo Scientific SuperBlock Blocking Buffers Guaranteed to be biotin free Our most popular blocking buffer SuperBlock Blocking Buffer now comes in both dry and liquid formats Many researchers have discovered that SuperBlock Blocking Buffer is the only blocking buffer needed for all of their applications Highlights e Fast blocking blocks ELISA plates in two minutes or membranes in five to 10 minutes e Non serum protein solution yields a high signal to noise ratio e Plates blocked with SuperBlock Blocking Buffer can be stored dry for up to 12 months e Liquid formulations available in PBS or TBS To order call
128. ples Clean Blot IP Detection Reagents are unique HRP and AP conjugates that reveal your target protein allowing clear specific Western blot detection from immunoprecipitation IP experiments and tissue extracts without any interference from denatured IgG Figure 3 Whereas conventional secondary antibodies recognize both denatured and native IgG our new reagents bind to only native IgG Figure 4 So unmask your results by simply substituting the secondary antibody with Clean Blot IP Detection Reagents for clear Western blots Figure 5 Highlights e Versatile recognizes most native antibodies independent of the host species Table 6 e Compatible clear results with IPs performed using Protein A Protein G or anti IgG agarose beads and any blocking buffer e g milk BSA or Thermo Scientific SuperBlock or StartingBlock Blocking Buffers e Cost effective eliminates the need to immobilize IgG and purchase separate kits specific for the primary antibody species membranes can be stripped and reprobed when chemiluminescent substrate is used e Flexible use any HRP or AP substrate including chemiluminescent fluorescent or colorimetric substrates e Easy to use simply replace the conventional secondary antibody with the Clean Blot IP Detection Reagents in your Western blotting protocol e Unobstructed detection clear P Western blot results without interference from denatured IgG bands Our Clean Blot IP Detecti
129. ponent system Ordering Information Product Description Pkg Size 34150 Lumi Phos WB 100 ml Chemiluminescent Substrate Sufficient materials for 1 000 cm membrane References Capasso J M et al 2003 Proc Natl Acad Sci USA 100 6428 6433 Ha S A et al 2003 Mol Biol Cell 14 1319 1333 Liu R Y et al 2000 J Biol Chem 275 21086 21093 Tikhonov l et al 2003 J Virol 77 3157 3166 Dilution Range of Antibody Approximate Substrate Product Measurement Color From 1 mg ml stock Sensitivity Enzyme Pierce ECL Substrate 32106 425 nm chemiluminescent 1 1 100 1 5K 10 pg HRP 2 1 1K 15K SuperSignal West Pico Substrate 34080 425 nm chemiluminescent 1 1 1K 1 5K 1 pg HRP 2 1 20K 100K SuperSignal West Dura Substrate 34075 425 nm chemiluminescent 1 1 1K 1 50K 250 fg HRP 2 1 50K 250K Supersignal West Femto Substrate 34095 425 nm chemiluminescent 1 1 5K 1 100K 1 fg HRP 2 1 100K 500K Lumi Phos Substrate 34150 440 nm chemiluminescent 1 1 200 1 2K 15 pg AP 2 1 5K 1 25K Pierce TMB Blotting Substrate 34018 Dark blue PPT 1 1 500 1ng HRP Lee 2 1 2K 20K Pierce 4 CN Substrate 34012 Blue purple PPT 1 1 500 1 ng HRP 2 1 2K 20K Pierce CN DAB Substrate 34000 Black PPT 1 1 500 1 ng HRP 2 1 2K 20K Pierce DAB Substrate 34001 Brown PPT 1 1 500 1 ng HRP 2 1 2K 20K Pierce Metal Enhanced DAB Substrate 34065 Brown black PPT 1 1 500 20 pg HRP 2 1 2K 20K Pierce BCIP Substrate 34040 B
130. preparation conditions prior to blotting of the protein have not destroyed antigenicity of the sample Caution Some proteins cannot be run under reducing conditions Insufficient binding to membrane e Adding 20 methanol to the transfer buffer helps binding Low MW antigen may pass through the membrane Use a membrane with a smaller pore size Insufficient amount of antibodies e Increase antibody concentrations Antibody may have poor affinity for the protein of interest e Antibody may have lost activity Perform a dot blot to determine activity Antibody concentrations are too high s Using too much primary or secondary antibodies can cause the signal to fade quickly which appears as a weak signal Insufficient amount of antigen present Load more protein onto the gel The antigen is masked by the e Try different blocking buffers blocking buffer e Optimize blocking buffer protein concentration Buffers contain sodium azide e Sodium azide is an inhibitor of HRP do not use sodium azide as a preservative in buffers Exposure time is too short e Lengthen the film exposure time Note SuperSignal Chemiluminescent Substrates will continue to glow for at least six hours Substrate incubation is too short e A five minute substrate incubation is required when using SuperSignal Substrates Inactive substrate e SuperSignal West Pico Chemiluminescent Substrate and SuperSignal West Dura Chemiluminescent Substrate are stable for up to 12 months a
131. press and purify GST PBD fusion proteins e Easy to use pull down conditions are optimized for immediate success even for first time users e Efficient simultaneous incubation of lysate GST PBD and glutathione resin in the spin column prevents sample loss e Validated each kit is functionally tested to ensure quality and performance e Sensitive optimized antibodies reagents and Western blotting procedure ensure accurate quantitative and reproducible results Kit Contents GTPase Pull down and Detection Kits contain sufficient materials to perform 30 pull down assays Currently certain kits differ in the format of glutathione resin that is included New lots of all kits will have the following component structure e GST Fusion Protein of Specific Binding Domain e Glutathione Agarose Resin 3 ml e GTPyS and GDP 100X 50 ul ea e Lysis Binding Wash Buffer 100 ml e GTPase Specific Primary Antibody 1 vial e SDS PAGE Sample Loading Buffer 2X 1 5 ml e Spin Columns and Collection Tubes Western blot using specific antibody Ordering Information Pkg Product Description Size 26185 Active Arf1 Pull Down and Detection Kit Kit 26186 Active Arf6 Pull Down and Detection Kit K 26187 Active RalA Pull Down and Detection Kit K 89854 Active Rho Pull Down and Detection Kit K 89855 Active Ras Pull Down and Detection Kit K 89856 Active Rac Pull Down and Detection Kit Ki 89857 Active Cdc42 Pull Down and Detection Kit Ki
132. ps and times required to complete a Western blot post transfer and before development with substrate using common classical methods the Pierce Fast Western reagent based system and instrument based system from Millipore Corporation Protocol Steps Classical Protocol Pierce Fast Western System Millipore SNAP i d Blocking Pretreatment 60 min Incubation with Primary 60 min 30 min 10 min Wash 45 min 1 min Incubation with Secondary 60 min 10 min 10 min Wash 45 min 15 min 1 min Total Time 270 min 55 min 22 min Key Limitations e Preparation of fresh non fat dry milk e For use with mouse or rabbit e Only 2 full sized blots per cycle blocking buffer each time primary antibodies Strips limited by the format of the blot holders available e Reliable vacuum source required Table 7 Western blot cost breakdown Western Blot Reagents Classical Protocol Pierce Fast Western Blot Protocol SNAP id System Protocol Blot holder NA NA 7 33 Membrane NC 2 48 2 48 2 48 Blocker 0 02 NA 1 32 Wash buffer TBST 1 48 NA 1 48 Secondary Antibody Conjugate 0 15 NA 0 15 Detection Substrate 1 43 NA 1 43 Film 5 x 7 1 01 1 01 1 01 Kit s price per blot NA 7 92 NA Total Cost Per Blot 6 57 11 41 15 20 Total Time for processing 4 5 hours 55 minutes 30 minutes or less Limitations e Preparation of fresh N A e Only 2 full blots per cycle Milk blocker e Optimization required e Hands on procedure e Vacuum source required e Requires
133. ption Pkg Size increase signal 37570 Protein Free TBS Blocking Buffer 1L Proprietary formulation in Tris buffered saline Traditional blocking buffers contain proteins that can cross react at pH 7 4 with Kathon Antimicrobial Agent with a system resulting in high background and reduced 37571 Protein Free 120 TBS Blocking Buffer 1L TS 2 Prote nef Bioekmna But TO Proprietary formulation in Tris buffered saline signa Igure 2 Fro eile ree 0G u utters e Iminate or l at pH 7 4 with 0 05 Tween 20 Detergent and Kathon minimize cross reactivity associated with protein based blocking Antimicrobial Agent buffers in ELISA Western blotting arrays and other 37572 T ble bier oe aoe S 1L roprietary formulation in phosphate burfered saline immunodetection applications at pH 7 4 with Kathon Antimicrobial Agent Hiahliahts 37573 Protein Free T20 PBS Blocking Buffer 1L Ightights Proprietary formulation in phosphate buffered saline e Protein free eliminate or minimize cross reactivity associated at pH 7 4 with 0 05 Tween 20 Detergent and Kathon Antimicrobial Agent with protein based blocking buffers e Compatible with multiple detection systems can be used in Western blots ELISA or arrays does not interfere with avidin biotin systems e High signal to background for optimal sensitivity e 1X formulation ready to use e Available with 0 05 Tween 20 Detergent already added saves time and money Protein Free Non
134. r a high level of performance regardless of the system you choose for your Western blotting or ELISA application Figure 3 They may be the only blockers you ever use Figure 3A 3B 3C 3D Nitrocellulose PVDF 30 minutes 24 hours Nitrocellulose PVDF 24 hours Full duration of SuperSignal West Dura Chemiluminescent Substrate light emission 1 2 3 4 5 1 2 3 4 B 3A 3B 1 2 3 4 5 1 2 3 4 5 3C 3D Figure 3A D Comparison of Thermo Scientific StartingBlock Blocking Buffer performance after stripping and reprobing Nitrocellulose vs PVDF when probed for the transferrin receptor CD71 Membrane Type Film Exposure Time 30 minutes Highlights Compatible with a wide range of detection systems e Works in both Western and ELISA applications e Rarely cross reacts with rabbit antibodies e Serum protein free e Biotin free Shorter blocking times e Western blotting 1 15 minutes e ELISA no wait blocking capability Strip and reprobe no reblocking necessary e Blots stay blocked with StartingBlock Blocker when our Restore Stripping Buffer Product 21059 is used allowing reprobing of the same blot without re blocking Superior signal to noise ratios in ELISA applications e Signal to noise ratios in the range of 10 1 20 1 have been realized with StartingBlock Blocking Buffer Ordering Information Product Description Pkg Size 37538 StartingBlock PBS Blocking Buffer 1L A protein based blocker f
135. r a wide variety of MW markers including unstained markers prestained markers and peroxidase conjugated markers Thermo Scientific Pierce Blue Prestained Molecular Weight Markers Room temperature stable markers are ready when you are 1 Open the plastic pouch and remove the Pierce Prestained Protein Molecular Weight Marker Mix which is packaged with a desiccant in a moisture resistant resealable pouch 2 Load 10 ul of DI water into a pipette tip puncture the foil over a single tube and dissolve the Pierce Prestained Markers 3 Dispense 5 10 ul of the marker into a sample well of the gel Each tube can be used for one or two gel lanes 4 Return the Pierce Prestained Marker Mix to its pouch and reseal The markers are stable at room temperature and can be kept on your bench top ready for your next gel References Foubert T R et al 2001 J Biol Chem 276 38852 38861 Prozialeck W C et al 2002 Infect Immun 70 2605 2613 Thermo Scientific Pierce 3 Color Prestained Markers Fresh marker every time with reference bands too B A Pierce Blue Pierce 3 Color Colorimetric and Colorimetric and MW of Chemiluminescent Chemiluminescent Chemiluminescent Detection using Component Detection on a BlueRanger Pierce In Gel Proteins Western Blot Proteins Detection Technology Myosin Phosphorylase B BSA Serum Albumin Ovalbumin Carbonic Anhydrase Trypsin Inhibitor Lysozyme Figure 1 Thermo
136. re storage at TC upon arrival Part B contains only the O GlcNAc specific monoclonal antibody This MAb is shipped on dry ice to ensure it maintains integrity during transit Upon its arrival store it at 20 C Thermo Scientific DyLight 549 649 Western Blotting Kit Fluorescent Western blotting made easy The fluorescent detection of two different targets on a single Western blot is easy to perform with the Thermo Scientific DyLight 549 649 Western Blotting Kit This highly optimized and convenient format saves you time and the frustration of having to evaluate reagents for compatibility with fluorescent Western blotting Table 8 The kit contains sufficient reagents for 10 Western blots and includes Pierce Dual Labeled Protein Molecular Weight Markers and secondary antibodies conjugated to DyLight 549 and 649 Fluorescent Dyes Highlights e DyLight 549 excitation emission maxima 560 574 nm e DyLight 649 excitation emission maxima 654 673 nm e Optimized format provides low background and high signal intensity Figure 10 e Convenient saves time and money associated with optimizing fluorescent Western blots For more information or to download product instructions visit www thermo com pierce Table 8 Recommended instruments for in gel and Western blot detection using Thermo Scientific DyLight Fluors Thermo Thermo Scientific Scientific Excitation Emission DyLight DyLight Company Instrument nm
137. rimary antibody The protocol requires minimal hands on time and yields results comparable to classic Western blotting with ECL The included Pierce ECL Substrate produces a chemiluminescent signal which is detected using photographic or other imaging methods Blots can be e Fast all the sensitivity of Pierce ECL substrate and saving 4 5 hours per blot e Convenient no expensive hardware or vacuum required no clogging issues repeatedly exposed to film to obtain optimal results or stripped of e Simple optimized protocol makes Western blot analysis easier the primary antibody and immunodetection reagents and reprobed than ever e Economical cost as little as 8 per blot no expensive About the Fast Western Blot Kits ECL Substrate consumables or extra equipment The Pierce Fast Western Blot Kit is reagent based system that e Excellent stability kit is stable for 1 year stored at 4 C provides optimized reagents for blocking antibody dilution and e Easy complete kit gives you all the components you need to detection of Western blots with Pierce ECL substrate The block and probe and develop a blot with your mouse or rabbit protocol is a quick efficient and economic way to obtain Western i l i primary antibody blot results without the hassle of buying an instrument and the Table 6 Comparison of common Western blotting protocols to the Pierce Fast Western System This table provides a side by side comparison of the ste
138. rmulation without inflated prices Paying twice what you should for an enhanced chemiluminescent ECL substrate For researchers interested in a quality product at a fair price there is a new option available Pierce ECL Western Blotting Substrate is an entry level Western blotting substrate that is value priced If you are currently using a needlessly expensive ECL substrate you can switch to Pierce ECL Western Blotting Substrate without any optimization Simply switch out the substrates and save a bundle Thermo Scientific Pierce ECL Reagent GE Healthcare Protein per well 40 50 kDa Amersham ECL Reagent Protein per well 40 50 kDa MW 4 2 1 0 5 0 25 ug MW 4 2 1 0 5 0 25 ug Marker Marker 1 5 minute exposure 1 5 minute exposure Thermo Scientific Pierce ECL Substrate Western blot detection of actin beta from HeLa cell lysate Dilutions of HeLa cell lysate were prepared and separated by electrophoresis The proteins were transferred to nitrocellulose membranes Product 88025 Membranes were blocked with 5 skim milk and then incubated with Mouse Anti Human Actin US Biological Swampscott MA at 1 pg ml The membranes were washed and then incubated with 0 2 ug ml of HRP conjugated Goat Anti Mouse IgG Product 31430 and then washed again Working solutions of the substrates were prepared according to the manufacturers instructions and added to the membranes for 1 minute The membranes were placed in plastic sheet prot
139. ross Adsorbed Rabbit lgG H L min x Hn Sr Prot Rabbit IgG F ab Rabbit IgG Fc Rabbit IgG H L min x GtHnMsSh Sr Prot Rabbit IgG H L Rabbit IgG H L min x HnMsRt Sr Brot Rat IgG H L Rat IgG F ab Rat IgG Fc Rat IgM u Rat IgG H L Rat lgG H L min x Ms Sr Prot Sheep IgG H L Streptavidin NeutrAvidin Protein t See Table at the top of page 20 for the Key to Abbreviations tt Stabilized pre diluted format also available see our web site Source Unconjugated Biotin Peroxidase Alk Phos Fluorescein Rabbit 3104 31720 31501 Mouse 31107 3730S 30400 81512 Rabbit 3008 31732 31402 31300 31509 Rabbit 3a 8040381553 Rabbit 9099 80483 81338731533 RabbitF ab 31109 8802 Goat 35 85D Rabbit 81587 Gott 8D Goat 31130 3770S 31490 81310 31829 Goat 31118 a Goat 30009 8774 am aa Goat 3m2 8B 81812 Goat 382 8 Goat 32 8 Goat 3098 _ _ _ 3m5 81575 Goat 324 37B Goat 3140 31417 31314 31577 Goat 31128 31782 Goat 3029 880 Goat i a E 8420 Mouse 30937 30784 3143 ee Rabbit 31042 31789 31423 31318 31535 GoatF ab 31638 GoatFlab pB _ __ d l GoatFlab S S 31899 Goat 369 Goat 3 Goat 31160 31800 31430 31320 31569 Goat Oo S S S k Goat 31164 31802 31432 31322 3154 Goat 31166 31803 31436 31324 31543 Goat 31968 31805 31437 31325 31547 Goat mmo 8048981827 Goat 3072 31804 31440 31326
140. rotein in the blocking buffer e Optimize blocking time and or temperature Block for at least 1 hour at RT or overnight at 4 C e Add Tween 20 Detergent to blocking buffer Use a final concentration of 0 05 Tween 20 Detergent Skip this step if you use StartingBlock T20 Blocking Buffer in PBS Product 37539 or TBS Product 37543 or SuperBlock T20 Blocking Buffer in PBS Product 37516 or TBS Product 37536 These buffers already contain Tween 20 Detergent at optimized concentrations e Prepare antibody dilutions in blocking buffer that contains 0 05 Tween 20 Detergent e Use a different blocking buffer e Do not use milk with avidin biotin systems Milk contains biotin e Test for cross reactivity Block a clean piece of membrane incubate with antibodies and then detect with SuperSignal Chemiluminescent Substrate e Reduce the concentration of the HRP conjugate e Increase number of washes and the volume of buffer used e Add Tween 20 Detergent to wash buffer if it s not already included Use a final concentration of 0 05 Tween 20 Detergent Caution If the concentration of Tween 20 is too high it can strip proteins off the membrane Skip this step if you use StartingBlock T20 Blocking Buffer in PBS Product 37539 or TBS Product 37543 or SuperBlock T20 Blocking Buffer in PBS Product 37516 or TBS Product 37536 These buffers already contain Tween 20 Detergent at optimized concentrations e Reduce the time the b
141. rrent Figure 1 Electrophoretic transfer We offer a wide selection of the most commonly used membranes for Western blotting including nitrocellulose and polyvinylidene difluoride PVDF At this stage before proceeding with the Western blot it is often desirable to stain all proteins on the membrane with a reversible stain to check the transfer efficiency Although the gel can be stained to determine if protein left the gel this does not ensure efficient binding of protein on the membrane Ponceau S stain is the most widely used reagent for staining proteins on a membrane However it has limited sensitivity does not photograph well and fades with time Pierce Reversible Stain is a superior alternative for staining protein on nitrocellulose Product 24580 or PVDF Product 24585 membranes Pierce Reversible Stain detects low nanogram levels of protein is easily photographed does not fade with time and takes less than 30 minutes to stain photograph and erase Thermo Scientific Pierce Fast Transfer System The new Pierce Fast Transfer System reduces protein transfer time from SDS PAGE to a membrane from 45 minutes to only 10 minutes The system consists of two parts the Pierce Fast Semi Dry Transfer Buffer and the Pierce Fast Semi Dry Blotter Use our cost effective Transfer System to obtain membranes that are Western blotting ready in 10 minutes without the need to purchase special consumables Fast tra
142. s Chromogenic Substrates Thermo Scientific Chemiluminescent Substrates 36 43 34 35 Pierce ECL Substrate 37 SuperSignal Chemiluminescent 38 42 Substrates and Kits Lumi Phos Chemiluminescent Substrate 43 Quick Reference Substrate Guide 43 Fast Western Blotting Kit 44 Specialized Western Blotting Kits 45 48 Thermo SuperSignal West Pico HisProbe Kit 45 Pierce O GLcNAc Western Blotting Detection Kit 46 Thermo Scientific DyLight 549 649 46 47 Western Blotting Kit Thermo Scientific DyLight 680 800 Near Infrared 47 Western Blotting Kit Thermo Scientific Active GTPase Pull Down 48 and Detection Kits Far Western Blotting 49 In Gel Western Detection 50 51 Thermo Scientific Pierce In Gel 51 Chemiluminescent Detection Kits Step 7 Film Thermo Scientific CL XPosure Film 52 Step 8 Stripping Buffer Optimizing the Signal to Noise Ratio 53 58 Protocol for Stripping an Immunoblot 54 Thermo Scientific Restore 55 Western Blot Stripping Buffers Thermo Scientific Restore PLUS 56 Western Blot Stripping Buffers Thermo Scientific Pierce 57 58 Background Eliminator Troubleshooting Guide 59 69 Blotting with Chemiluminescence 59 Optimizing Antibody Concentration 60 63 Problem Guide 64 67 Full Length Western Blotting Protocol Using 68 69 Chemiluminescent Substrates Recommended Reading 70 The term blotting refers to the transfer of biological samples from a gel to a membrane and their subsequent detection on the surface of the membra
143. s US 198 Primary Primary antibody Primary antibody Primary antibody Savings Antibody Reduction cost blot vs cost per 20 blots cost savings including cost of Pierce Factor PAR Factor PAR Factor AES treated vs untreated AES treated vs untreated Antibody Extender NC 0 Untreated 460 0 0 3X 153 40 306 60 198 60 12X 38 20 421 80 313 80 18 40 441 60 333 60 t Assumptions 1 Analysis based on 20 blots using an 8 cm x 10 cm nitrocellulose membrane 2 Pierce Antibody Extender NC 500 ml treats 20 blots 3 Primary antibody cost based on US 1 15 per ug 4 1 500 primary antibody dilution from a 1 mg ml stock 2 ug ml with an ECL Substrate 5 10 ml of primary antibody solution used per blot 6 20 ug of primary antibody used per untreated blot Thermo Scientific Pierce Antibody Extender NC Use Pierce Antibody Extender NC when vs Pierce Western Blot Signal Enhancer e You want a costly primary antibody to last as long as possible e You have plenty of target but the detection antibody is available Which one should you use ae In limited amount Pierce Antibody Extender NC and Pierce Western Blot Signal Enhancer are mutually exclusive i e you cannot extend your l antibody and increase signal at the same time so you can use Use Pierce Western Blot Signal Enhancer when only one of these products e You have a low abundance of target protein antigen but adequate primary antibodies with which to detect it
144. s used e Cross reactivity of secondary anti body is eliminated e Double probing is easily achieved using different labels on primary antibodies from the same host e Immunoreactivity of the primary antibody may be reduced as a result of labeling e Labeling a primary antibody for each target protein is time consuming and expensive e There is no flexibility in choice of primary antibody label from one experiment to another e Minimal signal amplification Table 2 Indirect detection method Advantages Disadvantages e Cross reactivity may occur with the secondary antibody resulting in nonspecific binding e Sensitivity is increased because each primary antibody contains several epitopes bound by the labeled secondary antibody which amplifies the signal e A wide variety of labeled secondary antibodies are available commercially e An extra incubation step is required in the procedure e Because many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection it is versatile e Immunoreactivity of the primary antibody is maintained because it is not labeled e Different detection markers can be used with the same primary antibody To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor SDS PAGE Separate protein sample by electrophoresis e Pierce SDS PAGE Sample Prep Kit
145. s without removing transferred proteins thereby allowing multiple reprobes of the target Thermo Scientific Restore PLUS Buffer Supplier A Supplier C 1 2 3 1 2 3 1 2 3 Original Actin Stripped Actin Actin Reprobed Cyclophilin B Figure 4 Reprobing with different antibodies HeLa cell lysate was probed for actin and detected with Pierce ECL Substrate Original panel Blots were then stripped with either Restore PLUS Stripping Buffer or competitive strip ping buffers Stripped panel The blots were then re blocked and reprobed for cyclophilin B and detected with Pierce ECL Substrate Reprobed panel STEP STEP Formul Wash Buff Highlights Ready and easy to use e No dilution necessary e No offensive odors e Store at room temperature Compatible with commonly used Western blotting reagents and other materials e Use on nitrocellulose and PVDF membranes stored wet or dry e Works with blocking buffer enzyme conjugate and chemilumi nescent substrate of choice Cost effective e Save valuable time and samples e Strip blots effectively the first time Robust but gentle e Transferred proteins remain viable e Strip the same blot up to five times Flexible e Strip and re probe to optimize antibody concentrations e Strip and re probe for new antigen of interest Figure 4 Ordering Information Product Description Pkg Size 46428 Restore PLUS Western Blot 30 ml Stripping Buffer Suffic
146. sely affect protein function High quality ethylene glycol is superior to glycerol for this purpose Thermo Scientific Pierce Peroxidase Stabilizer is an anti freeze solution that provides the highest purity and performance for antibodies conjugated with horseradish peroxidase HRP The most convenient option is to store antibodies at the working concentration so that dilutions do not have to be repeated for each use This is rarely possible with typical buffers but Thermo Scientific Guardian Peroxidase Conjugate Stabilizer Diluent provides this sort of protection for antibodies conjugated with horseradish peroxidase Antibodies can be stored at 1 1 000 ng ml for more than six months at room temperature without losing activity For pure enzymes and other non antibody proteins use the Protein Stabilizing Cocktail for storage and preservation Thermo Scientific Antibody Stabilizer and Storage Solutions Product Description Page 29810 Ethylene Glycol 50 solution 200 ml 21 31503 Pierce Peroxidase Conjugate Stabilizer 25 ml 21 37548 Pierce Peroxidase Conjugate Stabilizer Diluent 21 37552 Pierce Peroxidase Conjugate Stabilizer Diluent 1 L 21 For more information or to download product instructions visit www thermo com pierce Enzyme Substrates Ordering Information Specificit Chicken IgY H L Goat IgG H L min x HnMsRb Sr Prot Goat lgG H L Goat IgG F ab Goat IgG Fc Goat IgG H L min
147. ssume a starting concentration of 1 mg ml 1 Prepare dilutions of the protein sample in either TBS or PBS The proper dilution will depend on the antigen concentration present in the sample but because the concentration of the antigen of interest often is not known it is necessary to test a wide range of dilutions SuperSignal West Pico Substrate has picogram level detection sensitivity so sample dilutions can range from the low microgram to low picogram levels If too much antigen is applied the results may have any or all of the following detection of nonspecific bands blurred banding patterns and rapid signal deterioration 2 Prepare membranes The number of membrane pieces needed depends on how many different dilutions of primary and or sec ondary antibody will be screened Typically one or two dilutions of the primary antibody are tested with two or three different dilutions of the secondary antibody For example 1 1 000 primary with 1 50 000 secondary 1 1 000 primary with 1 100 000 secondary 1 5 000 primary with 1 50 000 secondary and 1 5 000 primary with 1 100 000 secondary 3 Place membranes on a paper towel Dot antigen dilutions onto the membranes Apply the smallest possible volume to the membranes 2 5 ul works well because the greater the volume that is applied the more diffuse the signal will be Allow the antigen dilutions to dry on the membranes for 10 30 minutes or until no visible moisture remains STEP
148. stern blot This dilution range is often appropriate for blots detected with a relatively insensitive chromogenic substrate but a much greater dilution is generally required for optimum performance with a sensitive chemiluminescent substrate such as SuperSignal West Chemiluminscent Substrates To view a comparison of our chemiluminescent substrates see Table 4 on page 42 Table 1 Advantages of enhanced chemiluminescence Sensitive e Intense signal with low background e Requires less antigen and antibody Fast e Rapid substrate processing of blot e Signal generated within seconds Stable e Unlike radioisotopes the shelf life is long e Store at room temperature or 4 C Hard copy results e Results are captured on X ray film e No fading or tearing of brittle membrane over time e Permanent record Film results e Signal output continues for a long time i e 8 24 hours e Can expose blot to film multiple times e Can optimize the developing method Can reprobe the blot e Can remove nonisotopic probes from the membrane e Can repeat immunodetection Large linear response e Can detect a large range of protein concentrations Quantitative e The X ray film can be scanned using a reflectance densitometer or using an imaging device such as a CCD camera For more information or to download product instructions visit www thermo com pierce Thermo Scientific Pierce ECL Western Blotting Substrate A reliable ECL fo
149. t 10 ug ml 100X more dilute than typical 1 mg ml preparations eliminating the inaccuracies associated with two stage dilution schemes required with traditional conjugate preparations for use with chemiluminescent substrates and other high sensitivity detection methods The liquid formulation of each prediluted HRP conjugate is stable at 4 C eliminating the need to freeze stock solutions for storage Ordering Information Product Description Pkg Size 32430 Stabilized Goat Anti Mouse IgG 2 ml H L Peroxidase Conjugated 32460 Stabilized Goat Anti Rabbit IgG 2 ml H L Peroxidase Conjugated Poly HRP Conjugates Get the ultimate sensitivity in immunodetection techniques with Thermo Scientific Pierce Poly HRP Conjugates Thermo Scientific Pierce Poly HRP Conjugates are designed to deliver the highest sensitivity and low background in immunoassays where sample volume is limited or when the target molecule is present at low levels Pierce Poly HRP Conjugates are purified to remove unconjugated probe molecules that reduce signal intensity by competing for binding sites with horseradish peroxidase HRP conjugated molecules In addition these conjugates are free of HRP monomers that may lead to increased background signal Together these features provide consistent and reliable sensitivity and deliver higher sensitivity than conventional HRP and poly HRP conjugates without the need for additional signal amplification steps The Pi
150. t RT SuperSignal West Femto Chemiluminescent Substrate is stable for at least six months at RT e To evaluate the substrate activity prepare a small amount of working solution In a darkroom add a small amount of HRP conjugate A blue light should be observed If no glow is observed either the substrate or the HRP conjugate is inactive e Ensure that there is no cross contamination between the two bottles of substrate Contamination between the two substrate reagents can cause a decline in activity Membrane has been e There may be some antigen loss or denaturation during membrane stripping procedures stripped and reprobed Optimize stripping procedure e Reprobe only when necessary e Avoid repeated reprobing of the same membrane Digestion of antigen onthe membrane Blocking substance may have proteolytic activity e g gelatin Protein degradation from blot storage Prepare a new blot For more information or to download product instructions visit www thermo com pierce Enzyme Substrates Possible Causes Precautions Solutions Antibody concentrations are too high Reduce antibody concentrations SDS caused nonspecific binding e Wash blots after transfer to immobilized protein bands e Do not use SDS during immunoassay procedure Possible Causes Precautions Solutions Antibody concentrations are too high Reduce antibody concentrations Too much protein is loaded e Reduce the amount of protein loaded o
151. tavidin e Ex Em 480 545 and 565 nm and 578 nm e Fluorescently labeled streptavidin Applications e Immunoassay reagent when bound to biotinylated enzymes or when conjugated to enzymes e Blocking protein for biotin rich tissue sections use at 0 1 for inhibition of endogenous biotin e Can be used with biotinylated enzymes Product 29339 or 29139 e Histochemistry e Western blotting e Conti L R et al 2001 J Biol Chem 276 41270 41278 e Histochemistry e Western blotting e Harriman G R et al 1999 J Immunol 162 2521 2529 e Nielsen PK et al 2000 J Biol Chem 275 14517 14523 e Histochemical staining e Fluorescence activated cell sorting FACS e Histochemical staining e Fluorescence activated cell sorting FACS e Histochemical staining can be used in double staining methods e Fluorescence activated cell sorting FACS e Histochemical staining e Fluorescence activated cell sorting FACS e Histochemical staining e Fluorescence activated cell sorting FACS e Attaches streptavidin to oxidized carbohydrate residues on glycoproteins e gt 4 moles hydrazide mole streptavidin Streptavidin DyLight 488 Conjugated Ex Em 493 518 lt Intense emission Streptavidin DyLight 549 Conjugated Ex Em 560 574 provides superior Streptavidin DyLight 594 Conjugated Ex Em 593 618 Streptavidin DyLight 633 Conjugated Ex Em 638 658 less conjugate Streptavidin DyLight 649 Conj
152. tein bands e Compatible with Laemmli sample buffer e Compatible with standard mini gel tanks so there is no need to purchase new equipment e Stains quickly and with high sensitivity using coomassie and silver stains e Transfers quickly and efficiently to nitrocellulose and PVDF membranes for Western blotting e More resolving power than Novex Gels e Plastic lane dividers prevent sample cross contamination Gel Specifications Cassette size 10 cm x 8 5 cm x 4 5 mm Gel size 8 cm x 5 8 cm x 1mm Shelf life 12 months 4 C Running buffer Tris HEPES SDS Sample buffer Tris HCI SDS Migration Table Gel Percentage 8 10 12 4 20 8 16 wow a 67 Migration Distance A N 1 00 Compatible Gel Tanks Thermo Scientific Owl P8 Systems IBI Universal Protein System Hoefer Tall Mighty Small SE 280 EC 4 Cell Mighty Small SE 260 SE 250 and Bio Rad Mini PROTEAN II amp 3 miniVE SE 300 Daiichi Mini 2 Gel amp 6 Gel C B S Scientific MGV 302 402 Novex XCell and II Surelock GradiGel Mini 4 Cell STEP STEP Formul Wash Buff Ordering Information Percent of Sample Well Product Acrylamide Sample Wells Volume Pkg Size 25200 8 10 50 ul 10 gels 25201 10 10 50 ul 10 gels 25202 12 10 50 ul 10 gels 25203 8 16 10 50 ul 10 gels 25204 4 20 10 50 pl 10 gels 25220 8 12 30 ul 10 gels 25221 10 12 30 ul 10 gels 25222 12 12 30 ul 10 gels 25223 8 16 12 30 ul 10 gels 25224 4 2
153. th optimizing fluorescent Western blots D D D Ss D D S in ra 2 2 9 D D Im N c m mia 3 2 9O N N N DO O o o o o e N D Tubulin TNFa Figure 11 The Thermo Scientific DyLight 680 800 Western Blotting Kit provides low background and high signal in two color Western blot detection Proteins were separated in a 4 20 Precise Protein Gel and transferred to a low fluorescence PVDF membrane The membrane was blocked overnight in SEA BLOCK Blocking Buffer and target proteins were detected according to the manufacturer s protocol Membranes were imaged with the LI COR Odyssey Infrared Imaging System Tubulin was detected from the indicated quantity of HeLa cell lysate Purified TNFa was detected at the indicated quantity Ordering Information Product Description Pkg Size 22855 DyLight 680 800 Western Blotting Kit Kit Sufficient reagents for 10 Western blots Includes DyLight 680 Goat Anti Mouse IgG H L 120 ul DyLight 800 Goat Anti Rabbit IgG H L 120 pl Fluorescent Dual labeled Protein 30 pl MW Markers Wash Buffer 30X 200 ml SEA BLOCK Blocking Buffer 500 ml Low Fluorescence PVDF Transfer Membrane 10 each Complementary Product 22860 Low Fluorescence PVDF Transfer Membrane 0 2 um 7 cm x 8 4 cm 10 pkg t See patent information on inside back cover To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor STEP STEP SDS PAGE E
154. than streptavidin conjugates 2 mg Phosphatase Conjugated e Better signal to noise ratio in assay systems e 3 8 ug biotin bound mg conjugate 31006 NeutrAvidin Fluorescein Conjugated e Fluorescent labeled NeutrAvidin Biotin Binding Protein 5 mg e Absorption 490 nm Emission 520 nm e gt 2 moles fluorescein mole NeutrAvidin Protein 31007 EZ Link Maleimide Activated e Prepare NeutrAvidin conjugates of proteins peptides 5 mg NeutrAvidin Biotin Binding Protein e Reacts spontaneously with free sulfhydryls in the pH range of 6 5 7 5 e 4 8 moles maleimide mole NeutrAvidin Protein To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor DS PAGE Electro Trangrer Thermo Scientific Streptavidin Products Wide selection of conjugates for almost any biotin based assay Originally isolated from Streptomyces avidinii streptavidin is a tetrameric biotin binding protein that we produce and offer in recombinant form Compared to the native protein recombinant streptavidin is smaller that the native protein MW 53K and has a more neutral isoelectric point pl 6 8 7 5 Streptavidin is STEP Formul Wash Buff STEP carbohydrate free and much less soluble in water than avidin resulting in high binding affinity capacity and specificity for biotinylated molecules Streptavidin conjugates are useful for secondary detection in Western blotting ELISA and cell and
155. tific DyLight Labeled Highly Cross Adsorbed Secondary Antibodies Highly cross adsorbed secondary antibodies conjugated with DyLight Fluors for superior fluorescent cell imaging Thermo Scientific DyLight Labeled Highly Cross Adsorbed Secondary Antibodies show minimum cross reactivity and are suitable for multiplex experiments They are designed to reduce nonspecific background staining and offer high specificity low background and increased assay sensitivity With these conjugates incubation times are reduced and there is good lot to lot consistency Each highly cross adsorbed antibody conjugated to a DyLight Dye is tested for cross reactivity in plate based IgG binding assays They did not exhibit any cross reaction to human bovine horse rabbit swine or rat IgGs The degree of cross reactivity was determined by ELISA and was typically less than 1 of the desired signal Our goat anti mouse conjugates showed low levels of crossreactivity with rat IgG however similar levels of cross reactivity were observed in other commercially available conjugates The affinity purified goat antimouse IgG H L was solid phase adsorbed to minimize cross reaction with human bovine horse rabbit and swine serum proteins while the affinity purified goat anti rabbit IgG H L was solid phase adsorbed to minimize cross reaction with human mouse and rat serum proteins To order call 800 874 3723 or 815 968 0747 Outside the United States contact yo
156. tion are also stabilized and buffered at approximately 1 mg ml Storage of stock solutions in 50 ethylene glycol or frozen in single use aliquots can greatly extend the shelf life of secondary antibodies See page 21 for additional information about these accessory products Selected secondary antibodies have been further purified to minimize cross reactivities to serum proteins of other species This purification is accomplished by adsorbing the antigen purified secondary antibody sample to a secondary affinity column containing immobilized serum proteins of selected species In the following product tables these antibody products that have been pre adsorbed to species serum proteins are indicated by the code min x Sp Sr Prot where particular species are specified according to the Species Key Table 3 Table 2 Comparison of AP and HRP enzymes Alkaline Horseradish Phosphatase Peroxidase Size 140 kDa 40 kDa Price Relatively Relatively Expensive Inexpensive Stability Unstable at lt 0 C Stable at lt 0 C Storage Number of Few Many Substrates Kinetics Slower Rapid pH optimum 8 10 5 7 Affinity Purified Secondary Antibodies The choice of secondary antibody also depends upon the type of label that is desired Many different labels can be conjugated to antibodies Radioisotopes were used extensively in the past but they are expensive have a short shelf life offer no improvement in signal to noise ratio and require spec
157. transfer method that is used most commonly for proteins is electroelution or electrophoretic transfer because of its speed and transfer efficiency This method uses the electrophoretic mobility of proteins and involves placing a protein containing polyacrylamide gel in direct contact with a piece of nitrocellulose or other suitable protein binding support and sand wiching this between two electrodes submerged in a conducting solution Figure 1 When an electric field is applied the proteins move out of the gel and onto the surface of the membrane where the proteins become tightly attached The resulting membrane Is a copy of the protein pattern that was in the polyacrylamide gel Transfer efficiency can vary dramatically among proteins based upon the ability of a protein to migrate out of the gel and its propen sity to bind to the membrane under Buffer Tank 9 particular set of conditions The efficiency of transfer el Transfer Membrane Gel Membrane Filter Sandwich anode ae 11 1 G w Electrodes depends on factors cathode e such as the com rection 0 O40 Sea ET position of the gel whether there is complete contact of the gel with the membrane the position of the electrodes the transfer time size and composition of proteins field strength and the presence of detergents Optimal transfer of proteins is generally obtained in low ionic strength buffers and with low electrical cu
158. trate generated signal should last for up to eight hours The blot can be re exposed to film or an imaging device as needed to obtain the optimal results Longer exposure times may be necessary as the blot ages If optimal results are not achieved repeat this procedure using different antigen and or antibody dilutions To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor SDS PAGE Electro Trangrer STEP STEP STEP Formul Wash Buff p R AL LAR BR RL R M oao EEEE T E Bo E IE I e N ee ear rinan a an Zp e Pe nife apaa H a8 TTA d O l Z P gt ae T l l E E yp N l l l d g l i Bi 1 LTA l a 1 i ACRAS Sen V 9 8 T 9 HLR L S ww kd gw W PF twee High Possible Causes Antibody concentrations are too high Incompatible blocking buffer was used Insufficient blocking of nonspecific sites Cross reactivity of antibody with other proteins in blocking buffer Insufficient washing Exposure time is too long Membrane problems Contamination or growth in buffers Precautions Solutions e The primary and or secondary antibody can cause high background if the concentrations used are too high e Decrease antibody concentrations e Compare different blocking buffers e Optimize blocking buffer The best blocking buffer is system dependent e Increase the concentration of p
159. uch enzyme is by far the primary cause of problems with a chemiluminescent Western blot It is essential that you adhere to the substrate manufacturer s dilution instruc tions not the antibody manufacturer s instructions when determining antibody titer because most substrates require different concentration levels 120 Too much enzyme B Appropriate enzyme ee Intensity S 40 20 Time Figure 1 A signal that peaks and terminates quickly is usually caused by the use of too much enzyme D D The following is a list of several indicators of too much enzyme 1 Inconsistent signal length a No signal signal fades before it can be detected by an imaging system b Signal terminates quickly c System gives inconsistent signal length from day to day i e It worked great yesterday but not very well today syndrome 2 Reverse image on the film dark background with clear or ghost band where the protein of interest is expected A ghosting of bands where the protein of interest is expected could be caused by using too much enzyme 3 Brown bands on the membrane where the protein of interest is expected The appearance of brown bands where the protein of interest is expected could be caused by the use of too much enzyme 4 High background and or unwanted bands Primary Antibody Primary Antibody 1 500 1 5 000 Secondary Antibody Seco
160. ugated Ex Em 654 673 lt Completely stable Streptavidin DyLight 680 Conjugated lt Ex Em 692 712 from pH 4 9 Streptavidin DyLight 750 Conjugated Ex Em 752 778 Streptavidin DyLight 800 Conjugated Ex Em 777 790 sensitivity and requires e Used to create immunoassay reagents e Localize glycoproteins on blot transfers followed by detection with a biotinylated enzyme e Fluorescence microscopy e Flow cytometry e Western blotting e ELISA e High content screening and other array platforms For more information or to download product instructions visit www thermo com pierce Pkg Size 1 mg 5 mg 100 mg 1 mg 2 mg 5 mg 1 mg 3 mg 1 mg 1 mg 1 mg 1 ml 0 5 ml 2 mg 1 mg 1 mg 1 mg 1 mg 1 mg 1 mg 1 mg 1 mg 1 mg Enzyme Substrates Thermo Scientific Avidin Products Convenient conjugates for assay detection Avidin is a tetrameric glycoprotein MW 67K purified from chicken egg white The highly specific interaction of avidin with biotin makes it a useful tool in designing nonradioactive detection systems The extraordinary affinity of avidin for biotin K 10 M allows biotin labeled molecules to be detected with excellent sensitivity and specificity References Chaiet I and Wolf F J 1964 Arch Biochem Biophys 106 1 5 Savage M D et al 1992 Avidin Biotin Chemistry A Handbook Rockford Illinois Pierce Chemical Company Wilchek M and Bayer E A 19
161. ulose membrane Pure GFP 6xHis tagged protein and E coli bacterial GFP 6xHis tagged lysate were separated by SDS PAGE Novex 10 20 Tris Glycine gels Gels were transferred to nitrocellulose membrane using the Bio Rad Mini Gel Transfer Unit Following the transfer the protein left in the gel was detected using the Thermo Scientific Pierce system with a 1 500 dilution of anti Penta His antibody followed by a 1 250 dilution of HRP labeled goat anti mouse antibody Lanes 1 5 E coli bacterial GFP 6xHis tagged lysate diluted 1 100 1 250 1 1 000 1 2 000 and 1 4 000 respectively Lanes 6 13 Pure GFP 6xHis tagged protein at 12 5 6 25 3 12 1 56 1 0 0 5 0 1 and 0 05 ng respectively Lane 14 6xHis tagged ladder 1 16 dilution Thermo Scientific Pierce In Gel Chemiluminescent Detection Highlights e Uniform representation of antigen s not skewed by inefficient transfer e Compatible with stripping and reprobing protocols e Compatible with protein staining e Sensitive to 1 ng comparable to an ECL Substrate Benefits e Many proteins such as membrane proteins do not transfer well to membranes the Pierce In Gel Detection Method prevents any problems associated with incomplete transfer e When transferring to membranes low MW proteins transfer more efficiently than higher MW proteins often skewing results e Transfer units buffers membranes and filter paper are eliminated e Procedure can be optimized by stripping and reprob
162. uments but sensitivity may not be as good as it is with film e Imagers sometimes require longer exposure times than required by film to obtain similar images e Background is less of an issue in many of these instruments therefore higher antibody concentrations may be used to achieve the best image in the shortest exposure time e No darkroom is necessary when using imaging instruments The instruments have their own light proof boxes e Refer to the instrument manufacturer s instructions for more information on an individual instrument STEP Formulate Wash Buffets 7 CTED Thermo Scientific CL XPosure Radiography Film Save 65 75 on film Highlights e Up to one third the price of competitive products Table 1 e Provides the same detection sensitivity as other commercially available films Figure 1 e Available in 5 x 7 8 x 10 9 5 x 11 8 14 x 17 or 18 x 24 cm sheets in packages of 25 50 or 100 non interleaved sheets Reference Tikhonov l et al 2003 J Virol 77 3157 3166 Kodak X Omat Blue XB Film Kodak BioMax MIR 1 Film Thermo Scientific CL XPosure Film Figure 1 Thermo Scientific CL XPosure Film vs Kodak Film Three types of X ray film were tested using identical Western blotting conditions 2 blue 1 grey The results showed no appreciable difference between any of these films The only significant difference is the cost per sheet of film Table 1 Ta
163. ur local branch office or distributor Product Description 35500 35503 35508 35511 35513 35516 35519 35550 35553 35558 35561 35563 35566 35569 Goat Anti Mouse IgG H L Highly Cross Adsorbed DyLight 405 Conjugated 1 mg ml Goat Anti Mouse IgG H L Highly Cross Adsorbed DyLight 488 Conjugated 1 mg ml Goat Anti Mouse IgG H L Highly Cross Adsorbed DyLight 549 Conjugated 1 mg ml Goat Anti Mouse IgG H L Highly Cross Adsorbed DyLight 594 Conjugated 1 mg ml Goat Anti Mouse IgG H L Highly Cross Adsorbed DyLight 633 Conjugated 1 mg ml Goat Anti Mouse IgG H L Highly Cross Adsorbed DyLight 649 Conjugated 1 mg ml Goat Anti Mouse IgG H L Highly Cross Adsorbed DyLight 680 Conjugated 1 mg ml Goat Anti Rabbit IgG H L Highly Cross Adsorbed DyLight 405 Conjugated 1 mg ml Goat Anti Rabbit IgG H L Highly Cross Adsorbed DyLight 488 Conjugated 1 mg ml Goat Anti Rabbit IgG H L Highly Cross Adsorbed DyLight 549 Conjugated 1 mg ml Goat Anti Rabbit IgG H L Highly Cross Adsorbed DyLight 594 Conjugated 1 mg ml Goat Anti Rabbit IgG H L Highly Cross Adsorbed DyLight 633 Conjugated 1 mg ml Goat Anti Rabbit IgG H L Highly Cross Adsorbed DyLight 649 Conjugated 1 mg ml Goat Anti Rabbit IgG H L Highly Cross Adsorbed DyLight 680 Conjugated 1 mg ml Ordering Information Pkg Size 0 5 ml 0 5 ml 0 5 ml 0 5 ml 0 5 ml
164. ure e Saves antibody primary and secondary antibodies are used highly diluted so they can be used for more blots 500 000 400 000 300 000 200 000 Net Relative Intensity 100 000 0 GE Healthcare Amersham ECL System Thermo Scientific SuperSignal West Pico Substrate Figure 3 Enhanced light emission kinetics Thermo Scientific SuperSignal Substrate vs GE Healthcare Amersham ECL System Net relative intensity six hours after incubation is much greater for SuperSignal West Pico Substrate than for the ECL System Table 2 Cost comparisons between SuperSignal West Pico Substrate and competitors substrates Substrate Cost Comparison Membrane 10 x 10 7 96 TBS Wash Buffer 1 29 SuperBlock Blocking Buffer 4 86 Primary Antibody 3 35 Secondary Antibody 0 04 Substrate 4 00 Film 0 93 Total Blotting Cost 22 29 SuperSignal West Pico Substrate GE Healthcare Amersham Perkin Elmer Western ECL Substrate Lightning Substrate 10 20 7 96 1 29 1 29 4 86 4 86 33 46 6 70 0 56 0 56 7 13 6 12 3 54 2 91 61 04 30 37 Anti CD54 Product MA5407 500 ug was used at the substrate manufacturer s recommended starting dilution Costs are based on January 2007 U S list prices for an 8 x 10 cm mini gel following manufacturer s instructions 1 Using Thermo Scientific products where applicable 2 Using GE Amersham Biosciences products where applicable 3 Using Perk
165. us solution To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor STEP STEP SDS PAGE Electro Trangrer Featured Product Thermo Scientific DyLight 405 488 549 594 633 649 680 750 and 800 Conjugates Bright new alternatives to Alexa Fluor CyDye and LI COR Fluorescent Dyes Thermo Scientific DyLight Dyes have absorption spectra ranging from 400 nm to 770 nm Table 5 and match the principal output wavelengths of common fluorescence instrumentation They exhibit higher fluorescence intensity and photostability than Alexa Fluor CyDye and LI COR Dyes in many applications and remain highly fluorescent over a broad pH range pH 4 9 Additionally DyLight Dye water solubility allows a high dye to protein ratio without precipitation during conjugation Table 5 Spectral properties of Thermo Scientific DyLight Fluorescent Dyes Thermo Scientific Emission Color DyLight Dye Blue 405 Green 488 Yellow 549 Red 594 633 649 Near Infrared 680 750 800 Excitation and emission maxima in nanometers 4 nm tMolar extinction coefficient M cm STEP STEP Formul Blocki Wash Buff Highlights e Available conjugated to commonly used secondary antibodies streptavidin and NeutrAvidin Protein conjugated using a molar ratio dye protein optimized to provide excellent fluorescent intensity e Stable for 1 year at 4 C e Antibo
166. ver and reoptimize experiment conditions A431 cell lysate was electrophor esed on a 4 12 NuPage Gel Novex and transferred overnight to nitrocel lulose The membrane was blocked with SuperBlock Blocking Buffer in PBS Product 37515 for 1 hour and incubated with 1 25 ng ml of HRP labeled mouse anti phosphotyrosine PY20 for 1 hour After the membrane was washed for 30 minutes SuperSignal West Dura Substrate was added The blot was exposed to film for 10 seconds and resulted in a completely black image caused by the antibody cross reacting with the blocking buffer A Using the old option another gel was prepared to optimize assay conditions The proteins were transferred overnight and then the membrane was blocked with a 5 dry milk solution for 1 hour The blot was detected with 2 5 ng ml of anti phosphotyrosine PY20 HRP and SuperSignal West Dura Substrate The blot was exposed to film for 10 seconds This optimization required a two day proce dure B Using the new option the initial dark film A was treated with Pierce Background Eliminator to allow the band images to appear in 4 minutes C To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch office or distributor STEP STEP SDS PAGE Electro Trangrer 30 Before Thermo Scientific Pierce Background Eliminator 25 1 Minute 2 5 Minutes O 4 Minutes N C3 Relative Intensity 1 000 250 62 5 15 625 Biotinylate
167. water insoluble product The substrate is widely used for immuno chemical assays and techniques because the color produced by the formazan is linear and stable over a wide dynamic range Ordering Information Pkg Size 1 g powder Product Description 34035 Pierce Nitro Blue Tetrazolium Chloride BCIP has a molecular weight of 433 6 and hydrolysis by AP results in a blue purple precipitate BCIP can be used as a chromogenic substrate for both immunoblotting and immuno histochemical studies Ordering Information Product Description Pkg Size 34040 Pierce 5 Bromo 4 chloro 3 indolyphosphate 1 g powder p toluidine Salt An ideal system for blotting or staining applications with AP is the combination of NBT and BCIP Figure 1 Together they yield an intense black purple precipitate that provides much greater sensitivity than either substrate alone This reaction proceeds at a steady rate allowing accurate control of its relative sensitivity NBT BCIP characteristically produces sharp band resolution with minimal background Cl 0 Br II 0 P OH N OH BCIP 5 5 S dichloro indigo white Figure 1 Reaction of AP with BCIP and NBT Z x T nied tautomerism T NBT formazan Ordering Information Product Description Pkg Size 34042 Pierce NBT BCIP 250 ml 34070 Pierce NBT BCIP Plus Suppressor 100 ml To order call 800 874 3723 or 815 968 0747 Outside the United States contact your local branch
168. x Hn Sr Prot Hamster IgG H L Hamster IgG H L Horse IgG H L Human IgG H L Human IgG Gamma Chain Specific Human IgG H L min x BvHsMs Sr Proti Human IgG F ab Human IgG F ab 2 min x BvHsMs Sr Prot Human IgG Fc min x BvHsMs Sr Prot Human IgM Fc5u Human IgM u Human IgA a Human IgA IgG IgM H L Human Kappa Chain Human Lambda Chain Human IgG H L min x Ms Sr Proti Human IgG H L min x BvHsMs Sr Prot Human IgG H L Human IgG Fc Human IgG Fc Human IgG H L Human IgA IgG IgM H L Mouse IgA min x Hn Sr Brot Mouse IgA IgG IgM H L Mouse IgG H L Mouse IgG H L Highly Cross adsorbed Mouse IgG H L min x BvHnHs Sr Proti Mouse IgG F ab Mouse IgG Fc Mouse IgG Fc min x BvHnHs Sr Proti Mouse IgM u Mouse IgG IgM H L Mouse IgG IgM H L min x BvHnHs Sr Prot Mouse IgG Fey subclasses 1 2a 2b 3 min x BvHnRb Sr Prot Mouse IgG Fey subclass 1 specific min x BvHnRb Sr Prot Mouse IgG Fey subclass 2a specific min x BvHnRb Sr Proti Mouse IgG H L Mouse IgG H L Mouse IgG H L min x Hn Sr Prot Mouse IgG F ab 2 Mouse IgG Fc Mouse IgM u Mouse IgG IgM H L Mouse IgG H L min x BvHnHs Sr Proti Mouse IgM u Mouse IgM u min x BvHnHs Sr Prot Mouse IgG IgM H L min x BvHnHs Sr Prot Rabbit IgG H L min x BvChGtGuHaHnHsMsRtSh Sr Proti Rabbit IgG H L Rabbit IgG H L Highly C
169. y MW Lumi Phos WB Substrate 1 200 1 2 000 or 0 5 5 0 ug ml 4 Transfer the protein from the gel to a membrane 7 Wash the membrane with wash buffer Use at least four to six Thermo Scientific Substrate Recommended Membrane changes of the wash buffer and as large a volume as possible Pierce ECL Substrate SuperSignal West Pico Substrate SuperSignal West Femto Substrate SuperSignal West Dura Substrate Lumi Phos WB Substrate Nitrocellulose or PVDF Nitrocellulose or PVDF Nitrocellulose or PVDF Nitrocellulose or PVDF Nitrocellulose For each wash suspend the membrane in wash buffer and agitate for at least 5 minutes Increasing the wash buffer volume and or the number of washes might reduce background Tris buffered saline TBS phosphate buffered saline PBS or another suitable wash buffer can be used Including a final concentration of 0 05 Tween 20 to the wash buffer may also help reduce background Note 1 Briefly rinsing the membrane in wash buffer before incubation will increase the efficiency of the wash step Note 2 If using an enzyme conjugated primary antibody proceed directly to Step 10 For more information or to download product instructions visit www thermo com pierce 8 Thermo Scientific Substrate Pierce ECL Substrate Enzyme Substrates Incubate the blot with enzyme conjugated secondary antibody or avidin for 1 hour with shaking at RT For recommended antibody or avidin conjug
170. y conditions is best reperforming the gel electrophoresis process to test each new primary antibody or antibody concentration is time consuming and expensive You can forget about starting over when you use Restore Western Blot Stripping Buffer Optimize assay conditions Using Pierce SuperSignal West Substrates the secondary antibody concentrations are optimized after a single stripping and re probing cycle Figure 2 Test different primary antibodies There s no need to waste precious sample and re run a gel to test different primary antibodies Simply strip the membrane with Restore Stripping Buffer to remove the antibodies It takes only 5 15 minutes depending on the affinity of the primary antibody After stripping re probe with a new primary antibody and detect with SuperSignal Chemiluminescent Substrate Figure 3 Figure 2 Antibody optimization study Western blots of Interleukin 2 diluted 20 0 156 ng were detected using SuperSignal West Pico Chemiluminescent Highlights e Saves time no need to re run gels e Saves precious sample re probe the membrane using the same target sample e Provides efficient removal proprietary formulation works better than homemade buffers e Gentle formulation does not damage target protein after stripping and re probing e Odor free no mercaptans means no acrid odors e Economical less expensive than other competing stripping buffers References Baolin Z
171. y made in the wash buffer containing a blocking agent The presence of a small amount of blocking agent and detergent in the antibody diluent often helps to minimize background We offer a wide variety of labeled secondary antibodies for use in Western blotting The labels include biotin fluorescein rhodamine DyLight Dyes horseradish peroxidase and alkaline phosphatase For the complete list of labeled secondary antibodies please refer to pages 25 26 CTED STEP Wash Buff fs Validated Primary Antibodies for Western Blot Applications There are thousands of antibodies commercially available how ever many of them don t function well The new Thermo Scientific Intracellular Pathway Antibodies eliminate the need to screen numerous antibodies to find one that detects your target in Western blot applications Our antibodies have been validated by sIRNA mediated knockdown of the target protein using Thermo Scientific Dharmacon ON TARGET plus SMARTpool siRNA to ensure target detection Additionally each antibody is vigorously optimized for Western blot applications using the Thermo Scientific SuperSignal Chemiluminescent Detection Module Product 82200 Our scientists are dedicated to providing the highest quality reagents and have screened gt 500 antibodies to 185 different targets Table 1 Some of the commercially available antibodies we screened did not detect the specified protein Figures 1 and 2 Instead of spen

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