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Western Blotting Handbook and Troubleshooting Guide

1. 30 minutes Chemilmager 4000 50 2512 56 3 3 1 1 6 0 8 0 4 0 2 0 1 05 03 013 006 003 ng aassas Highlights e 24 hour light emission 10 times longer than that offered by other enhanced chemilumines cent 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 Save your antibody antibodies can be diluted much further when using SuperSignal West Dura Extended Duration Substrate than with other chemiluminescent substrates perform 25 50 times more blots than possible with other chemiluminescent substrates e Comes with HRP labeled secondary antibodies e ntense signal that is generated immediately and easily detected on film or chemilumines cent imager systems e Working solution is stable for at least 24 hours the kit itself is stable for at least one year with ambient shipping conditions PRODUCT DESCRIPTION PKG SIZE 34075 SuperSignal West Dura 100 ml Extended Duration Substrate Includes Luminol Enhancer 50 ml Stable Peroxide Buffer 50 ml HRP Conjugated Goat Anti Rabbit 1 ml HRP Conjugated Goat Anti Mouse 1 ml 34076 SuperSignal West Dura 200 ml Extended Duration Substrate Includes Luminol Enhancer 100 ml Stable Peroxide Buffer 100 ml HRP Conjugated Goat Anti Rabbit 1 ml HRP Conjugated Goat Anti Mouse 1ml 37071 SuperSignal West Dura Extended 2
2. V Py 3X E wu A A gt t T n i nandbook and rag ue Re Featuring the SuperSignal West Family of Products 5 Table of Contents 0 1 oo ee ee AU 1 Western Blotting Overview PR 2 3 Transfer Protein to a Membrane 20 eller 4 MemCode M CIRCITER 4 5 Blocking Nonspecific Binding Sites ee eee 6 blu bcm dB SEV NNEUT LAXE et 6 masier UC Ue e a a e E aa TELAM DET 6 Blocking Buffer Optimization SERERE 7 OG E E TETTE ie e A a A aa a a a M a ns 8 9 Washing the Membrane 99 3 7 a EM omes an eee s Sr e Wats 10 WasmbBullers 2 v oe eom Ln eubetun E Ue GE a TOURS 10 Primary and Secondary Antibodies 0 0 00 cee eee ees 11 Conjugate Stabilizer Solutions eee eee 12 Affinity Purified Secondary Antibodies 13 15 Labeling Your Own Antibodies EE 16 MMO BUTE ENZVINCS OT ae hak St aad aes 17 EZ Link Activated Enzymes 000 cee e eee e aes 18 19 EZ Label Fluorescent Labeling Kits Le 20 Optimizing Antibody Concentration EE 21 22 Chromogenic Subsitat s es eo a LA S l 23 24 Chemiluminescent Substrates 0 000 cece eee ee eee 25 31 SuperSignal Chemiluminescent Substrates 26 30 LumiPhos Chemiluminescent Substrate 31 Quick Reference Substrate Guide 0 002 e ee eee 31 Lae iA 5
3. SuperSignal West Pico Substrate Use any nitrocellulose 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 o PRODUCT DESCRIPTION PKG SIZE 34080 SuperSignal West Pico 500 ml Chemiluminescent Substrate Includes Luminol Enhancer 250 ml Stable Peroxide Buffer 250 ml 34077 SuperSignal West Pico 100 ml Chemiluminescent Substrate Includes Luminol Enhancer 2 x 25 ml Stable Peroxide Buffer 2 Xx 25 ml 34079 SuperSignal West Pico 50 ml Chemiluminescent Substrate Trial Kit Includes Luminol Enhancer 25 ml Stable Peroxide Buffer 25 ml 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 conjugated Anti Rabbit IgG Anti Mouse IgG or NeutrAvidin Biotin Binding Protein e SuperBlock Blocking Buffer e TBS Wash Buffer e SuperSignal West Pico Substrate PRODUCT DESCRIPTION PKG SIZE 34081 SuperSignal West Pico Complete Kit Mouse IgG Detect
4. 20 in PBS Product 37516 or TBS Product 37536 e SuperBlock Blocking Buffer Blotting in PBS Product 37517 and in TBS Product 3 537 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 STEP 4A Formulate Wash Buffers Choose a buffer e Phosphate Buffered Saline PBS Product 28372 e Tris Buffered Saline TBS Product s 28376 and 28379 e Modified Dulbecco s PBS Product 28374 e Carbonate Bicarbonate Buffer Packs Product 28382 e MES Buffered Saline Product 28390 e BupH Borate Buffer Packs Product 28384 e BupH Citrate Carbonate Buffer Pack Product 28388 Western Blotting the Pierce Way Enzyme Substrates Add the detection reagent Formulate Wash Buffers SUS ae Add detergent to blocking wash buffers to reduce nonspecific binding Chemiluminescent Substrates e SuperSignal West Pico Chemiluminescent substrate Product s 34077 and 34080 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 SuperSignal West Femto Maximum Sensitivity Substrate Product s 34096 and 34095 e Supe
5. PRODUCT DESCRIPTION 24580 MemCode Reversible Kit Protein Stain Kit for Nitrocellulose Membranes Sufficient material to stain protein and reverse the stain from 10 8 cm x 8 cm nitrocellulose membranes Includes MemCode Reversible Stain A broad spectrum stain for proteins transferred to nitrocellulose membranes MemCode Destain 1 000 ml Enhances protein band detection by eliminating background stain MemCode Stain Eraser Reverses protein band staining on demand 24585 MemCode Reversible Protein Kit Stain Kit for Polyvinylidene Difluoride Membrane Sufficient material to stain protein and reverse the stain from 10 8 cm x 8 cm PVDF membranes Includes MemCode Sensitizer PVDF membrane pre treatment agent MemCode Reversible Stain A broad spectrum stain for proteins transferred to PVDF membrane MemCode Destain Enhances protein band detection by eliminating background stain MemCode Stain Eraser Reverses protein band staining on demand Reagent grade methanol required but not supplied supplements the Destain and Stain Eraser formulations 250 ml 500 ml 250 ml 250 ml 1 000 ml 500 ml MemCode Reversible Protein Stain Protocols A Nitrocellulose Membrane Staining Protocol Wash membrane with ultrapure HO 2 Add MemCode Stain Shake 30 seconds Protein bands appear turquoise in color Destaining Protocol Add MemCode Destain Shake 5 minute
6. Western Blot Stripping Buffer Strip time off your research with Restore Stripping Buffer Tired of re running electrophoresis gels and waiting to see your results Although optimiz ing assay conditions is the best way to achieve optimum results re performing 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 new Restore Western Blot Stripping Buffer Optimize Assay Conditions Using Pierce SuperSignal West Substrates Figure 27 shows how the secondary antibody concentrations are optimized after a single stripping and re probing cycle A chemilumines cent detection system is the most sensitive method to detect any reagent still bound after the stripping procedure Test Different Primary Antibodies There s no need to waste precious sample and re run a gel to test different primary antibod ies Simply strip the membrane with Restore Stripping Buffer to remove the first primary antibody and set of reagents It takes only 5 15 minutes depending on the affinity of the pri mary antibody After stripping re probe with a new primary antibody Figure 28 shows how the SuperSignal Chemiluminescent Detection System can analyze two different proteins using the same blot Figure 27 Antibody optimization study Western blots of Interleukin 2 diluted 20 0 156 ng were detected using SuperSignal West Pico Chemilu
7. e Biotinylation Kits Product 21059 e Protein A Protein G and Protein L labeled with fluorescein e IgG Elution Buffer Product s 21004 rhodamine HRP AP or biotin and 21009 e Avidin Streptavidin and NeutrAvidin Biotin Binding Protein labeled with fluorescein rhodamine HRP or AP e Secondary antibodies labeled with fluorescein rhodamine HRP AP or biotin Transfer Protein to a Membrane Following electrophoresis the protein must be transferred from the electrophoresis 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 The 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 to transfer them from the gel to the matrix Electrophoretic transfer of proteins involves placing a protein containing polyacry lamide gel in direct contact with a piece of nitrocellulose or other suitable protein binding Support and sandwiching this between two electrodes submerged in a conducting solution Figure 2 When an electric field is applied the proteins move out of the polyacrylamide gel and onto the surface of the membrane where the proteins become tightly attached The resulting membrane is a copy of the protein pattern t
8. e Enhances Western blot detection e All components are room temperature stable Table 1 Comparison of MemCode Reversible Protein Stain with Ponceau S Ponceau S Reversible Stain e Weak binding low sensitivity general protein stain e Detection limit 250 ng e Red bands are difficult to photograph e Stained protein bands fade within hours e Typical staining time 9 minutes MemCode Reversible Protein Stain e Tight binding higher sensitivity general protein stain e Detection limit 25 50 ng e Turquoise blue bands are photographed easily e Turquoise bands do not fade over time but they can be reversed Typical staining time 60 seconds e Background eliminated quickly with low pH wash 12 3 4 12 3 4 A MemCode B Ponceau S Stain Stain 123 45 6 7 8 910 A MemCode Stain Figure 3 MemCode 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 Both gels were electroblotted to nitrocellulose membrane Blot A was treated with MemCode Stain for 30 seconds and destained according to the protocol Blot B was stained with 0 196 Ponceau stain for 5 minutes and destained The blot stained with MemCode Stain demonstrates superior visual detection of bands GST Lysate loading volumes Lane 1 3 Lane 1 5 yl Lane 2 10 ul Lane 3
9. 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 For more product information or to download a product instruction booklet visit www piercenet com Featured Product UnBlof amp 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 ECL Substrate Benefits e Many proteins such as membrane proteins do not transfer well to membranes the use of UnBlot Technology prevents any problems associated with incomplete transfer e When performing transfers low molecular weight 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 reprobing 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 on the gel 12 34 56 7 Membrane Detection ih Detection Figure 24 High sensitivity using UnBlot Detection Pure GFP 6xHis tagged an
10. Enhances detection of targets transferred to either nitrocellulose or PVDF independent of membrane pore size e Works with the most commonly used Western blot ting 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 capa bility of your specific analyte 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 PRODUCT DESCRIPTION PKG SIZE 21050 Qentix Western Blot Kit Signal Enhancer Sufficient reagent for ten 10 cm x 10 cm blots Includes Enhancer Reagent 1 250 ml Enhancer Reagent 2 250 ml Figure 29 Enhanced chemiluminescent detection of identical serial dilutions of IL 6 before and after treatment with Qentix Western Blot Signal Enhancer Untreated blot Blot treated with Qentix Western Blot Enhancer 1 2 3 4 5 6 T Figure 30 Enhanced chromogenic detection of identical serial dilutions of IL 6 before and after treat ment with Qentix Western Blot Signal Enhancer Ultrapure 1 Ultrapure 2 Ultrapure amp UH A HO yp amp 0 Start your N J J detection d Nw J uu j E E l o j protocol 1 Rinse membrane after 2 Incubate membrane with 3 Rinse memb
11. For the complete list of labeled secondary antibodies please refer to pages 13 15 Antibody Labels 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 special handling Alternative labels are biotin fluorophores and enzymes The use of fluorophores requires fewer steps however special equipment is needed to view the fluorescence Also a photograph must be taken if a permanent record of the results is desired Enzymatic labels are used most commonly and although they require extra steps they can also be extremely sensitive Alkaline phosphatase AP and horseradish peroxidase HRP are the two enzymes that are used extensively as labels for protein detection An array of chromogenic fluorogenic and chemiluminescent substrates is available for use with either enzyme For a detailed compari son of these two enzymes see Table 2 Alkaline phosphatase a 140 000 dalton protein that is generally isolated from calf intestine cat alyzes 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 a
12. H L Rabbit 31109 31302 Fragment of min x Hn Sr Prot 0 5 mg 0 5 ml Host Antibody Anti GUINEA PIG Guinea Pig IgG H L Goat AN mg Anti HAMSTER Hamster IgG H L Goat 31115 31750 1 5 mg 1 5 mg Hamster IgG H L Rabbit 31120 31587 31652 2 mg 1 5 mg 1 5 mg Anti HORSE Horse IgG H L Goat 31116 31760 2m 1 5m Anti HUMAN Human IgG H L Goat 31130 31770 31529 31656 31410 31310 2 mg 1 5 mg 2 mg 2 mg 2 ml 1 ml Human IgG Goat 31118 Gamma Chain Specific 0 5 mg Human IgG H L Goat 31119 31774 31531 31412 Human IgG F ab Goat 31122 31312 2m 1 ml Human IgG F ab Goat 31132 31414 Human IgG Fc Goat 31123 31416 min x BvHsMs Sr Prot 1 5 mg 1 5 ml Human IgM Fc5p Goat 31136 31575 31415 2 mg 2 mg 2 ml Human IgM p Goat 31124 31778 0 5 mg 0 5 mg Human IgM Fc5j Goat 31138 min x Bv Sr Prot 1 5m Human IgA o Goat 31140 31577 31417 31314 2 mg 2 mg 2 ml 1 ml Human IgG IgM Goat 31134 31776 H L 2m 2 ml Human IgA IgG Goat 31128 31782 31418 31316 IgM H L 2 mg 2 ml 2 ml 1 ml Human Kappa Chain Goat 31129 31780 0 5 m 0 5 m Human Lambda Chain Goat 31131 0 5 mg Human IgG H L Mouse 31135 31420 min x Ms Sr Prot 2m 1 5 ml See Table 3 on page 12 for the Key to Abbreviations Tel 800 874 3723 or 815 968 0747 www piercenet com Affinity Purified Secondary Antibodies Product Pkg Size Specificity Description Host Unconj Biotin LC Fluorescein Rhodamine Peroxidase Alk Phos Anti HUMAN Human IgG H
13. L Mouse 31137 31784 continued min x BvHsMs Sr Prot 1 5 mg 1 ml Human IgG H L Rabbit 31143 31786 2 mg 1 5 ml Human IgG H L Rabbit 31147 min x Ms Sr Prot 1 5 mg Human IgG Fc Rabbit 31142 31789 31535 31423 31318 2 mg 1 5 ml 1 5 mg 1 5 ml 1 ml Human IgM Fc Rabbit s J 5i 4 0 5Z0 UC o ooo Anti HUMAN Human IgG Fc Goat 31163 F ab Fragment 1 mg of Host Antibody Human IgG H L Goat oe mg 2 o ooo Human IgA IgG Goat 31539 IgM H L 1 mg Human IgG Mouse 31155 min x MsBvHs Sr Prot 1 5 mg Anti MOUSE Mouse IgA o Goat 31169 min x Hn Sr Prot 1 mg Mouse IgA IgG Goat 31171 IgM H L 2 mg Mouse IgG H L Goat 31160 31800 31569 31660 31430 31320 2 mg 2 ml 2 mg 2 mg 2 ml 1 ml Mouse IgG H L Goat 31164 31802 31541 31661 31432 31322 min x BvHnHs Sr Prot 1 5 mg 1 5 mg 1 5 mg 1 5 mg 1 5 ml 1 ml Mouse IgG F ab Goat 31166 31903 31543 31436 31324 2 mg 2 ml 2 mg 2 ml 1 ml Mouse IgG Fc Goat 31168 31905 31547 31663 31437 31325 2 mg 2 ml 2 mg 2 mg 2 ml 1 ml Mouse IgG Fc Goat 31170 31439 31327 min x BvHnHs Sr Prot 1 5 mg 1 5 ml 1 ml Mouse IgM y Goat 31172 31904 31992 31662 31440 31326 2 mg 0 5 mg 2 mg 2 mg 2 ml 1 ml Mouse IgM p Goat 31176 31585 31664 min x BvHnHs Sr Prot 1 5 mg 1 5 mg 1 5 mg Mouse IgG IgM Goat 31182 31907 31586 31444 31328 H L 2m 2 ml 15m 2 ml 1 ml Mouse IgG IgM Goat 31184 31446 31330 H L min x BvHnHs Sr Prot 1 5 mg 1 5 ml 1 ml Mouse IgG H L Horse 3
14. Phosphate Buffered 40 pack Saline Packs BupH Tris Buffered Saline Great wash buffer for Western blots Each pack yields 500 ml of 25 mM Tris 0 15 M NaCl pH 7 2 when dissolved in 500 ml deion ized water 10 pack makes 5 liters total 40 pack makes 20 liters total PRODUCT DESCRIPTION PKG SIZE 28380 BupH Tris Glycine 40 pack Buffer Packs 28376 BupH Tris Buffered 40 pack Saline Packs 28379 BupH Tris Buffered 10 pack Saline Packs Surfact Amps 20 Purified Detergent Solution Specially purified form of Tween 20 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 PRODUCT DESCRIPTION PKG SIZE 28320 Surfact Amps 20 6 x 10 ml Primary and Secondary Antibodies 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 anti bodyresource com or www sciquest com on the Internet Alternatively a primary antibody may be made to recognize the antigen of interest For more information on producing a custom antibody see the Antibody Production and Purification technical section of the Pierce Technical Handbook and Catalog Both polyclonal and monoclonal antibodies
15. S list prices and there is no need to purchase additional enhancers for nitrocellulose membranes Long signal duration allows you to redevel op blots over and over e Attomole range detection and crystal clear dint background e Immediate strong signal no more waiting 15 to 30 minutes for the signal to become Figure 19 Serial dilutions of recombinant mouse IL 2 were separated electrophoretically on a 4 20 strong enough to detect SDS polyacrylamide gel The separated protein was then transferred to nitrocellulose membrane followed by blocking The membranes were subsequently incubated in a 1 500 1 ug ml dilution of purified rat anti Ready to use no mixing required with this mouse IL 2 followed by a 1 5 000 200 ng ml dilution of AP labeled goat anti rat IgG The membranes one component system were washed and then incubated in Lumi Phos WB Substrate for five minutes prior to film exposure PRODUCT DESCRIPTION PKG SIZE Lumi Phos WB Chemiluminescent Substrate overcomes all of the limitations posed by 34150 Lumi Phos WB 100 mi conventional chemiluminescent substrates for AP Chemiluminescent Substrate Lumi Phos WB Substrate provides sensitivity in the low picogram range enabling you to detect mere attomoles of your target ligand Lumi Phos WB Substrate also produces less References background noise than other popular chemiluminescent substrates for AP providing a better Capasso J M et al 2003
16. an avidin biotin system Milk contains biotin 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 e 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 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 binding 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 Tel 800 874 3723 or 815 968 0747 www piercenet com Blotting with Chemiluminescence Troubleshooting Guide Weak Signal or No Signal Possible Causes Precautions Solutions Proteins did not transfer properly e After transfer is complete stain the gel with a total protein stain to determine tr
17. biotin Although superBlock Blocking Buffer Product 7 37515 often gives excellent results we recommend testing several blocking reagents for their suit ability in a particular system There is no blocking reagent that will be the optimal reagent for all systems As shown in Figure 6 various proteins were analyzed by Western blotting to determine the optimal blocking condition for nonspecific sites Recombinant Human Cyclin B1 Wild Type p53 and Mouse fos Baculovirus lysates were diluted in Lane Marker Reducing Sample Buffer 1 50 1 10 or 1 2 and separated electrophoretically on a 12 SDS polyacrylamide gel The proteins were transferred to nitrocellulose membrane and cut into strips The membrane strips were blocked for 1 hour at RT with shaking in Blocker Casein in TBS 1 BSA in TBS SuperBlock Blocking Buffer in TBS or 5 non fat milk in TBS Tween 20 0 05 was added to all blocking buffers The membranes were then incubated with the appropriate primary antibody at 0 5 ug ml prepared in the different blocking solutions for one hour at RT with shak ing Each membrane strip was washed with TBS followed by a one hour incubation in HRP con jugated Goat anti Mouse antibody prepared in the different blocking buffers at a 25 ng ml con centration The membranes were washed with TBS A working solution of SuperSignal West Pico Chemiluminescent Substrate was prepared and added to each membrane for 5 minutes The membranes w
18. of peroxides and carbonyl in a 10 solution e Enhances signal to background ratio PRODUCT DESCRIPTION PKG SIZE 28320 Surfact Amps 20 Purified 6 x 10 ml Detergent Solution Tel 800 874 3723 or 815 968 0747 www piercenet com Washing the Membrane Like other immunoassay procedures Western blotting consists of a series of incubations with different immunochemical reagents separated by wash steps Washing steps are nec essary to remove unbound reagents and reduce background thereby increasing the signal to noise ratio Insufficient washing will allow high background while excessive wash ing 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 physiologic buffer such as Tris buffered saline TBS or phosphate buffered saline PBS without any additives More commonly a deter gent such as 0 05 Tween 20 Product 28320 is added to the buffer to help remove nonspecifically bound material Another common technique is to use a dilute solution of the blocking buffer along with some added detergent Including the blocking agent and adding a detergent in wash buffers helps to minimize background in the assay For best results use high purity detergents such as Surfact Amps Detergents for Western blotting BupH Dry Buffers The most advanced versatile time
19. 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 Do not handle the membrane with bare hands Always wear clean gloves or use forceps e Use a new membrane e Incubate membranes separately to ensure that membrane strips are not covering one another during incubations Tel 800 874 3723 or 815 968 0747 AG www piercenef com Full Length Western Blotting Protocol Using Chemiluminescent Substrates 1 Make the protein solution of interest in a sample buffer and heat it to boiling for 5 minutes The sample buffer Should contain the following e 03 M TriseHCl e 5 SDS to denature the protein and to generate a constant anionic charge to mass ratio for the denatured protein chains e 50 glycerol to give the sample a higher density than the running buffer allowing the Sample to sink to the bottom of the well e A low M W dye for dye front determination e As needed a reducing agent such as 100 mM B mercaptoethanol dithiothreitol or TCEP that will reduce the disulfide bonds present in the protein sample Adjust solution to pH 6 8 2 Add the protein solution in the sample buffer to an SDS polyacrylamide gel SDS PAGE 3 Separate the proteins electrophoretically by M W 4 Transfer the protein from the gel to a membrane SuperSignal West SuperSignal West SuperSignal West Lumi Phos Substrate Pico Substra
20. or 815 968 0747 www piercenet com Labeling Your Own Antibodies Chemical cross linking reagents have become an invaluable tool in the scientific community These reagents are used in preparing antibody enzyme conjugates and other labeled protein reagents After the protein is conjugated to an appropriate enzyme it may then be used as a detection reagent in a variety of assays and applications A number of cross linking methods have been used to prepare enzyme conjugates For example an hydroxysuccinimide ester can be prepared from a ligand of interest then reacted with a primary amine on the surface of the enzyme While this method is necessary in some applications such as those in which the ligand does not contain a primary amine it is not useful as a general purpose method Antigen binding site Light Chains VH NH Fab Fab Hinge NH Region S S Papain S Pepsin Carbohydrate CH Carbohydrate Fc S S NH CH CH Heavy Chains NH Amines on lysine residues Sulfhydryls created when antibody is reduced For more product information or to download a product instruction booklet visit www piercenet com Antibody Modification Sites Antibodies can be easily modified to contain labels such as biotin fluorescent tags or enzymes to create reagents for Western blot ting ELISA immunohistochemical staining and in vivo targeting Pierce of
21. protein sample Makes it easy to confirm atypical results When immunoblot results are not expected reprobing allows the use of the same pro tein sample without going back to gel electrophoresis Makes it easy to correct mistakes Immunoblotting requires many steps providing ample opportunity for mistakes to occur By stripping the membrane the blot can be reused instruction booklet visit www piercenet com For more product information or to download a product Following any stripping procedure the blot Should be tested to ensure that all of the detec tion reagents were removed The membrane should be washed several times with blocking agent incubated with secondary antibody then reincubated with chemiluminescent substrate If the primary antibody was effectively removed by the stripping procedure no secondary anti body should bind to the membrane and no Signal should be produced If bands are still vis ible 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 strip ping buffer or change methods entirely Note 1 Optimization of both incubation time and temperature is essential for best results Note 2 If the blot cannot be stripped immediately after chemiluminescent detection the blot can be stored in PBS at 4 C until the stripping procedure is t
22. saving buffer product line available The ultimate in convenience 1 Reach for the sealed foil pack sitting conveniently on your bench top 2 Open pour into beaker and add water 3 The fresh buffer is ready to use in practical aliquots so there s no waste The ultimate in versatility 1 Routine buffers are designed for use in Western blotting dialysis cross linking ELISAs immunohistochemistry protein plate coating biotinylation and other applications 2 Using one buffer source maintains consistency and eliminates variables within the lab The ultimate in integrity 1 BupH Buffers are protected from contamination and are fresh every time 2 Carry out applications with confidence in buffer quality 3 Test assured with the Pierce 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 preparation tracking or refrigeration hassles 3 Move forward with your work by eliminating re tests due to buffer problems instruction booklet visit www piercenet com For more product information or to download a product BupH Phosphate Buffered Saline Packs Great wash buffer for Western blots Each pack yields 500 ml of 0 1 M phosphate 0 15 M NaCl pH 7 0 when dissolved in 500 ml deionized water 20 liters total PRODUCT DESCRIPTION PKG SIZE 28372 BupH
23. tions examine interaction sequences using synthetic peptides as probes and identify protein protein interactions without using antigen specific antibodies with this method of analysis Far Western blotting vs Western blotting The far Western blotting technique is quite similar to Western blotting In a Western blot an antibody is used to detect the corresponding antigen on a membrane In a classical far Western analysis a labeled or antibody detectable bait protein is used to probe and detect the target prey protein on the membrane The sample usually a lysate contain ing the unknown prey protein is separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS PAGE or native PAGE and then transferred to a membrane When attached to the surface of the membrane the prey protein becomes accessible to probing After transfer the membrane is blocked and then probed with a known bait protein which usually is applied in pure form Following reaction of the bait protein with the prey pro tein a detection system specific for the bait protein is used to identify the corresponding band Table 10 Specialized Far Western Analysis By creative design of bait protein variants and other controls the far Western blotting method can be adapted to yield very specific information about protein protein interactions For example Burgess et al used a modified far Western blotting approach to determine sites of contact among sub
24. washes may reduce background Tris buffered saline TBS phosphate buffered saline PBS or another suitable wash buffer can be used The addition of 0 05 Tween 20 to the wash buffer may also help reduce background Note 1 Briefly rinsing the membrane in wash buffer prior to incubation will help increase the efficiency of the wash step Note 2 If using an enzyme conjugated primary antibody proceed directly to Step 10 For more product information or to download a product instruction booklet visit www piercenet com Protocol continued 8 Incubate the blot with enzyme conjugated secondary antibody or avidin for 1 hour with shaking at RT For recom mended antibody or avidin conjugate 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 SuperSignal West SuperSignal West SuperSignal West Lumi Phos Pico Substrate Femto Substrate Dura Substrate WB Substrate Recommended 1 20 000 1 100 000 1 100 000 1 500 000 1 50 000 1 250 000 1 5 000 1 25 000 Secondary Antibody or 10 50 ng ml or 2 0 10 ng ml or 4 0 20 ng ml or 40 200 ng ml Dilutions from 1 mg ml stock 9 Repeat Step 4 to wash away any unbound enzyme conjugated secondary antibody It is crucial to thoroughly wash the membrane after the incubation with the enzyme conjugate 10 If the working solution has not been prepared prepare it now F
25. water insoluble product The substrate is widely used for immunochemical assays and techniques because the color produced by the formazan is linear and stable over a wide dynamic range PRODUCT DESCRIPTION PKG SIZE 34035 Nitro Blue Tetrazolium Chloride 1 g powder BCIP has a molecular weight of 433 6 and hydrolysis by alkaline phosphatase results in a blue purple precipitate that can be deposited on nitrocellulose or nylon membranes BCIP can be used as a chromogenic substrate for both immunoblotting and immunohistochemi cal studies PRODUCT DESCRIPTION PKG SIZE 34040 5 Bromo 4 Chloro 3 indolyphosphate p toluidine Salt 1 g powder An ideal system for blotting or staining applications with AP is the combination of NBT and BCIP 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 little background staining of the membrane alkaline Br Ori OH phosphatase icio Oo HDD UU INI NBT formazan ete EP E dichloro indigo white Figure 14 Reaction of alkaline phosphatase with BCIP and NBT PRODUCT DESCRIPTION PKG SIZE 34042 1 Step NBT BCIP 200 ml 34070 1 Step NBT BCIP Plus Suppressor 100 ml For more product information or to download a product instruction booklet visit www pie
26. work well for Western blotting Polyclonal antibodies are less expensive and less time consuming to pro duce 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 prepa rations 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 order your FREE Affinity Purification Handbook from a Pierce Customer Service representative at 800 874 3723 or 815 968 0747 or from your local Perbio Science Branch office or 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 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 several different host Species and if a secondary antibody causes high background in a particular assay another host species may be chosen Another option to reduce background is to us
27. 0 ml Duration Substrate Trial Kit Includes Luminol Enhancer 10 ml Stable Peroxide Buffer 10 ml ECL Plus Substrate 9 minutes film 50 25 125 63 31 1 6 08 0 0 2 0 1 05 03 013 006 003 ng 15 minutes Chemilmager 4000 50 25 125 63 31 1 6 08 0 4 0 2 0 1 05 03 013 006 003 ng all Figure 17 50 ng of recombinant mouse IL 2 was serially diluted to 0 003 ng and electrophoresis was performed The gel to be used for SuperSignal West Dura Substrate was transferred to nitrocellulose membrane and the gel to be used for ECL Plus Substrate was transferred to PVDF membrane The membranes were blocked and then incubated with a 1 ug ml dilution of the primary antibody rat anti mouse IL 2 After washing the membranes were incubated with the secondary antibody HRP conjugated goat anti rat IgG The membranes were washed again and then incubated with substrates that were pre pared 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 and the ECL Plus Blot was exposed for 15 minutes The exposure of the ECL Plus Blot was not extended to 30 minutes due to the high background that had already accumulated at 15 minutes Tel 800 874 3723 or 815 968 0747 www piercenet com Featured Product SuperSignal West Femto Maximum Sensitivity Substrate SuperSignal West Femto Max
28. 095 SuperSignal West Femto 100 ml Maximum Sensitivity Substrate Sufficient substrate for 800 cm of blotting membrane Includes Luminol Enhancer Solution 50 ml Stable Peroxide Solution 50 ml HRP Conjugated Goat Anti Rabbit 1 ml HRP Conjugated Goat Anti Mouse 1ml 34096 SuperSignal West Femto 200 ml Maximum Sensitivity Substrate Sufficient substrate for 800 cm of blotting membrane Includes Luminol Enhancer 100 ml Solution Stable Peroxide Solution 100 ml HRP Conjugated Goat Anti Rabbit 1 ml HRP Conjugated Goat Anti Mouse 1 ml 34094 SuperSignal West Femto 20 ml Maximum Sensitivity Substrate Trial Kit Includes Luminol Enhancer 10 ml Solution Stable Peroxide Solution 10 ml References Adilakshmi T and Laine R O 2002 J Biol Chem 277 4147 4151 Conti L R et al 2001 J Biol Chem 216 41270 41278 Guo Y et al 2001 J Biol Chem 216 45791 45799 Featured Product Lumi Phos WB Chemiluminescent Substrate 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 noise ratios produce clearer blots A chemiluminescent substrate for alkaline phosphatase detection that provides the best of both worlds high sensitivity and low background th C C C C C C C C C C C C C C S eM al cu OS SEO UR ERN e Inexpensive less expensive than other sub uiri inc strates based on 2004 U
29. 1181 31806 1 5 mg 1 5 mg Mouse IgG H L Rabbit 31188 31810 31561 31665 31450 31329 2m 1 5 ml 1 5m 1 5m 1 5 ml 1 ml Mouse IgG H L Rabbit 31190 31812 31452 31334 min x Hn Sr Prot 1 5 mg 1 ml 1 ml 0 5 ml Mouse IgG F ab Rabbit 31192 31811 31559 31666 31451 31331 2m 1 5 ml 1 5m 1 5 mg 1 5 ml 1 ml Mouse IgG Fc Rabbit 31194 31913 31555 31455 31332 2 mg 1 5 ml 1 5 mg 1 5 ml 1 ml Mouse IgM y Rabbit 31196 31814 31557 31456 31333 2m 1 5 ml 1 5m 1 5 ml 1 ml Mouse IgG IgM Rabbit 31198 31815 31558 31457 31335 H L 2 mg 1 5 ml 1 5 mg 1 5 ml 1 ml Mouse IgG H L Goat 31185 31565 31438 min x BvHnHs Sr Prot 1m 1m 0 5 ml See Table 3 on page 12 for the Key to Abbreviations For more product information or to download a product instruction booklet visit www piercenet com Affinity Purified Secondary Antibodies Product Pkg Size Specificity Description Host Unconj Biotin LC Fluorescein Rhodamine Peroxidase Alk Phos Anti MOUSE Mouse IgM p Goat 31178 F ab Fragment 1 mg of Host Antibody continued Mouse IgM y Goat 31186 31442 min x BvHnHs Sr Prot 1 mg 0 5 ml Mouse IgG IgM Goat 31448 H L min x BvHnHs Sr Prot 0 5 ml Mouse IgG H L Rabbit 31189 1m Anti RABBIT Rabbit IgG H L Donkey 31821 31568 31458 31345 min x BvGtHnHsMsRtSh Sr Prot 0 5 ml 0 5 mg 0 5 ml 0 5 ml Rabbit IgG H L Goat 31210 31820 31670 31460 31340 2 mg 1 5 mg 2 mg 2 ml 1 ml Rabbit IgG H L Goat 31212 31822 31583 31462 31342 min x Hn Sr P
30. 15 yl Lane 4 BlueRanger Marker Mix Product 26681 10 ul 12345 6 7 8 910 B Ponceau S Stain Figure 4 Comparison of MemCode Reversible Protein Stain with Ponceau S stain on PVDF mem brane ColorMeRanger Unstained Protein M W 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 MemCode Stain for 1 minute and destained according to the pro tocol Blot B was stained with 0 1 Ponceau S in 5 acetic acid for 5 minutes and destained according to the published protocol Lane 10 BlueRanger Prestained M W Marker Mix Product 26681 12 3 4 1234 gt Control B MemCode Stain Figure 5 Immunoblot analysis of GST by chemiluminescent detection after MemCode Staining destaining and stain reversal Different amounts of purified GST protein were applied to two 10 Tris glycine SDS polyacrylamide gels Both gels were electroblotted to nitrocellulose membranes The con trol membrane Panel A was not treated with MemCode Reversible Protein Stain Panel B was subjected to the staining detaining and stain erasing protocol of the MemCode Kit Both membranes were then probed with anti GST incubated with goat anti rabbit IgG HRP conjugate and detected using Pierce SuperSignal West Dura Substrate Product 34075 Lane 1 125 pg Lane 2 250 pg Lane 3 500 pg and Lane 4 1 ng
31. 510 UnBlot In Gel Chemiluminescent Kit Detection Kit for Biotinylated Antibody Probes Includes Streptavidin HRP 0 1 mg Dilution Buffer 50 ml Phosphate Buffered Saline 17 packs 10 Tween 20 6x10 ml UnBlot Substrate 110 ml Cellophane Exposure Sheets 10 pack 33515 UnBlot In Gel Chemiluminescent Kit Detection Kit for GST Tagged Proteins Includes Anti Glutathione 0 25 mg S transferase GST HRP Dilution Buffer 50 ml Phosphate Buffered Saline 17 packs 10 Tween 20 6 x 10 ml UnBlot Substrate 110 ml Cellophane Exposure Sheets 10 pack 33550 UnBlot Chemiluminescent 110 ml Substrate 33499 Hands Off Incubation Colander 1 unit References Desai S Dworecki B and Cichon E 2001 Anal Biochem 297 94 98 Desai S Dworecki B and Cichon E 2002 Immunodetection of proteins within polyacrylamide gels Bioluminescence and Chemiluminescence World Scientific Publishing Co pp 413 416 Roberts K P et al 2002 Biol Reprod 67 525 533 Tel 800 874 3723 or 815 968 0747 www piercenet com 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 radio 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 o
32. Do not use milk to block membranes when using an avidin biotin system Milk contains biotin Test for cross reactivity Block a clean piece of membrane incubate with antibodies and then detect with SuperSignal Chemiluminescent Substrate Reduce the concentration of the HRP conjugate Insufficient washing e Increase number of washes and the volume of buffer used e Add Tween 20 to wash buffer if it s not already included Use a final concentration of 0 05 Tween 20 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 Exposure time is too long e Reduce the time the blot is exposed to film Membrane problems 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 e Do not handle membrane with bare hands Always wear clean gloves or use forceps Contamination or growth in buffers e Prepare new buffers For more product information or to downlo
33. GE i e denaturing conditions with or without a reducing agent offers more information about molecular weight presence of disulfides and subunit composition of a prey protein but may render the prey protein unrecognizable by the bait protein In these cases the proteins may need to be subjected to electrophoresis under native conditions i e nondenaturing and without reducing agent Transfer to Membrane After separation on the gel proteins are electrophoretically transferred from the gel to a membrane in two to 16 hours The type of membrane e g nitrocellulose or PVDF used for the transfer of proteins is critical as some proteins bind selectively or preferably to a partic ular membrane The efficiency and rate of protein transfer is inversely proportional to the molecular weight of the protein In some cases transfer conditions alter the shape of the protein and destroy or sterically hinder the interaction site on the protein For Far Western analysis it is essential that at least the interaction domain of the prey protein is not disrupt ed by the transfer or is able to re fold on the membrane to form a three dimensional 3 D structure comprising an intact interaction site Generally a significant percentage of the pro tein population renatures upon removal of SDS When SDS is eliminated during the transfer process transferred proteins generally renature with greater efficiency and are therefore more easily detected by far Western
34. ImmunoPure Alkaline Phosphatase 100 mg Maleimide activation The heterobifunctional cross linker SMCC Product 22360 and its water soluble analog Sulfo SMCC Product 22322 have more general utility in preparing immunologically active horseradish peroxidase or alkaline phosphatase conjugates They are most useful when preparing conjugates of reduced IgG and F ab because these meth ods involve the initial step of preparing a maleimide activated sulfhydryl reactive enzyme derivative Studies have shown that the two step maleimide method is superior to glu taraldehyde or metaperiodate methods for enzyme conjugation Figure 8 The maleimide method gives higher yields with less polymer ization producing a conjugate preparation with superior immunoassay characteristics Maleimide activated enzymes can be prepared using the heterobifunctional cross linker Sulfo SMCC This reagent contains an A hydroxy sulfosuccinimide Sulfo NHS functional group and a maleimide functional group and it is water soluble due to the presence of the sulfonate 504 group on the V hydroxysuc cinimide ring The sulfonate group also contributes to the stability of the molecule in aqueous solution A study of the hydrolysis rate of the maleimide functional group from Sulfo SMCC showed that it is less prone to hydrolysis to the maleamic acid than the non sulfonated SMCC The maleimide groups of Sulfo SMCC exhibit no decomposition at pH 7 at 30 C
35. PNAS 100 6426 6433 signal noise ratio and a clearer image Because signal generation is immediate there s no Ha S A et al 2003 Mol Biol Cell 14 1319 1333 need to wait 15 to 30 minutes for a measurable signal All of these benefits are combined Liu R Y et al 2000 J Biol Chem 275 21086 21093 with a low low price Tikhonov et al 2003 J Virol 77 3157 3166 Table 9 Quick reference substrate guide Dilution range of Antibody Approximate Substrate Product Measurement Color From 1 mg ml stock Sensitivity Enzyme 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 50K 60 fg HRP 2 1 100K 500K 1 Step TMB Blotting Substrate 34018 Dark blue PPT 1 1 500 1 ng HRP 2 1 2K 20K 1 Step 4 CN Substrate 34012 Blue purple PPT 1 1 500 1 ng HRP 2 1 2K 20K CN DAB Substrate 34000 Black PPT 1 1 500 1 ng HRP 2 1 2K 20K DAB Substrate 34001 Brown PPT 1 1 500 1 ng HRP 2 1 2K 20K Metal Enhanced DAB Substrate 34065 Brown black PPT 1 1 500 20 pg HRP 2 1 2K 20K Lumi Phos Substrate 34150 440 nm chemiluminescent 1 1 5K 15 pg AP 2 1 25K 1 Step NBT BCIP Substrate 34042 Black purple PPT 1 1 500 30 pg AP 2 1 2 5K 1 Step NBT BCIP Suppressor Substrate 34070 Black Purple PP
36. Set of portable proto cols that include step by step instructions for the most frequently used and essential techniques The protocols are printed on durable cards enabling them to be used easily at the bench This helpful guide along with high quality prod ucts from Pierce will help you purify immobilize label and store antibodies and perform common procedures such as immunoprecipitation Western blotting and ELISA 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 Affinity Purified Secondary Antibodies Product Pkg Size Specificity Description Host Unconj Biotin LC Fluorescein Rhodamine Peroxidase Alk Phos Anti BOVINE Bovine IgG H L Goat 31100 31710 2 mg 2 ml Bovine IgG H L Rabbit 31103 31712 2 mg 1 5 ml Anti CHICKEN Chicken IgY H L Rabbit 31104 31720 31501 31401 2 mg 1 5 ml 1 5 mg 1 5 ml Anti GOAT Goat IgG H L Donkey 31108 1 5 mg Goat IgG H L Mouse 31107 31730 31512 31400 min x HnMsRb Sr Prot 1 5 mg 1 ml 1 mg 1 ml Goat IgG H L Rabbit 31105 31732 31509 31650 31402 31300 2 mg 1 5 mg 1 5 mg 1 5 mg 1 5 ml 1 ml Goat IgG F ab Rabbit 31153 31753 31553 31403 31405 2 mg 1 5 ml 1 5 mg 1 5 ml 1 ml Goat IgG Fc Rabbit 31133 31733 31533 31433 31337 Anti GOAT F ab Goat IgG
37. T 1 1 500 30 pg AP 2 1 2 5K NBT Substrate 34035 Blue purple PPT 1 1 250 100 pg AP 2 1 2 5K BCIP Substrate 34040 Blue purple PPT 1 1 250 100 pg AP 2 1 2 5K Fast Red Substrate 34034 Red PPT 1 1 250 320 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 Tel 800 874 3723 or 815 968 0747 www piercenet com There are several methods for capturing data generated from chemiluminescent Western blots including X ray film cooled CCD cameras and phosphorimagers that detect chemilu minescence Cooled CCD cameras which offer the advantages of instant image manipulation higher sensitivity greater resolution and a larger dynamic range than film also eliminate the need for a darkroom and film processing equipment However this tech nology has one drawback it requires a substrate that produces an intense signal of long enough duration to be captured by the camera To meet the needs required by this new technology Pierce introduced SuperSignal West Dura Extended Duration Substrate Product 34075 Its 24 hour light emission and ultra intense signal combine to allow researchers to take full advantage of the benefits offered by digital imaging equipment Most instrument companies know and re
38. aa ee i NET TT 32 Oo gigs MTS T 32 Western Blot Detection Kit 0 2c eee eee 34 Far Western Blotting Sno sig S ALONSO PE 39 36 In Gel Western Detection mE 37 39 Optimizing the Signal to Noise Ratio 0 00 0 cee eee 40 Restore Western Blot Stripping Buffer 42 Qentix Western Blot Signal Enhancer 200000 43 Erase It Background Eliminator 0000 cee eee 44 45 Troubleshooting Guide Blotting with Chemiluminescence 46 49 Western Blotting Protocol Using Chemiluminescent Substrates 90 51 Recommended Reading 0 0c cece eee eee eee eee 52 For more product information or to download a product instruction booklet visit www piercenef com 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 membrane Western blotting also called immunoblotting 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 The specificity of the antibody antigen interaction enables a single protein to be identified in the midst of a com plex protein mixture Western blotting is commonly used to positively identify a specific protein in a complex mixture and to obtain qualitative and semiquantitative data about that protein The first step in a Western
39. abeled Protein Molecular Weight Marker Mix Product 26651 e TriChromRanger Prestained Protein Molecular Weight Marker Mix Product 26691 e ColorMeRanger Unstained Protein Molecular Weight Marker Mix Product 26671 Electro Transfer Transfer proteins to membrane e MemCode Reversible Protein otain Kit for Nitrocellulose Membranes Product 24580 and for PVDF Membranes Product 24585 e Tris Glycine Transfer Buffer Product 28380 Qentix Western Blot Signal Enhancer Product 21050 e Nitrocellulose Membrane 0 2 um Product s 77012 88013 and 88024 e Nitrocellulose Membrane 0 45 um Product s 77010 77011 88014 and 88025 e Capture PVDF Membrane 0 45 um Product s 88585 and 08518 For a detailed Western blotting protocol see pages 50 51 Protein Detection Made Easy For detection of proteins that cannot be efficiently transferred to a membrane Pierce has developed UnBlot Technology that allows positive identification of proteins directly in a gel Product zs 33500 33505 33510 and 33515 Blocking Block nonspecific sites e StartingBlock Blocking Buffer in PBS Product 37538 and in TBS Product 37542 v 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 in TBS Product 37535 e SuperBlock T20 Blocking Buffer Contains 0 05 Tween
40. ad a product instruction booklet visit www piercenet com o Blotting with Chemiluminescence Troubleshooting Guide High Background that is Blotchy or Speckled Possible Causes Precautions Solutions Antibody concentrations are too high 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 Use a fresh high quality conjugate Wrong blocking buffer was used Compare different blocking buffers Insufficient blocking of Optimize blocking buffer The best blocking buffer is system dependent nonspecific sites e Increase concentration of protein 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 to blocking buffer A concentration of 0 05 Tween 20 is recommended 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 Cross reactivity of antibody with Use a different blocking buffer other proteins in blocking buffer Do not use milk to block membranes when using
41. al 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 itself and on the type of enzyme conjugate to be used For example with applications using an alkaline phosphatase 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 interaction eliminating background altogether without altering or obscuring the epitope for antibody binding For true optimization of the blocking step for a particular immunoassay 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 important 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 exces Sive background staining and a reduced signal to noise ratio Using excessive concentrations of blocker may mask antibody antigen interactions or inhibit the marker enzyme again causing a reduction of the signal to noise ratio When developing any new immunoassay it is import
42. amine F ab frag ments 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 ImmunoPure Polyclonal Antibodies are purified by immunoaffinity chromatography using antigens coupled to agarose gels Affinity purification helps to eliminate nonspecific antibod ies resulting in an increase in sensitivity and specificity with a decrease in background The purification process involves an elution procedure yielding antibodies with high avidity These antibodies exhibit both maximal binding to antigens and minimal cross reactivity to other molecules Conjugated antibodies are affinity purified prior to the conjugation process selected ImmunoPure Antibodies have been further purified by passing them through immobilized serum proteins from other species This additional processing minimizes 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 Table 3 Key to abbreviations for individual species Bv Bovine Gu Guinea Pig Hs Horse Rt Rat Ch Chicken Ha Hamster Ms Mouse oh Sheep Gt Goat Hn Human Rb Rabbit oW swine ImmunoPure Polyclonal Conjugated Antibodies contain bovine serum albumin as a stab
43. ansfer efficiency to the membrane Note Total protein stains may not be able to detect low quantities of antigen e Use MemCode 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 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 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 M W antigen may pass through the membrane Use a membrane with a smaller pore size Insufficient amount of antibodies 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 Insufficient amount of antigen present Load more protein onto the gel The antigen is masked by the e Try different blocking buffers blocking buffer Optimize blocking buffer protein concentration Buffers co
44. ant 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 anti body antigen pair has unique characteristics If a blocking buffer that does not cross react with your system cannot be found UnBlot In Gel Protein Detection is an alternative choice The UnBlot System specifically detects proteins within the gel and requires no blocking see page 39 for more information Pierce offers a complete line of blocking buffers for Western blotting including BLOTTO Casein BSA SEA BLOCK and the exclusive SuperBlock and Starting Block Blocking Buffers For more product information or to download a product instruction booklet visit www piercenet com Featured Products 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 192 mM glycine at a pH of approximately 8 when dissolved in 400 ml deionized water and 100 ml of methanol 20 liters total 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 deion ized water 10 pack makes 5 liters total 40 pack makes 20 liters total 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 Tra
45. as been e There may be some antigen loss or denaturation during membrane stripping procedures stripped and reprobed Optimize stripping procedure Reprobe only when necessary Avoid repeated reprobing of the same membrane Digestion of antigen on the membrane Blocking substance may have proteolytic activity e g gelatin Protein degradation from blot storage Prepare a new blot For more product information or to download a product instruction booklet visit www piercenet com o Blotting with Chemiluminescence Troubleshooting Guide 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 onto the gel onto the gel Black blots with white bands or signal that decreases quickly 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 Problem Partly developed area or blank areas Possible Causes Precautions Solutions Incomplete transfer
46. b Highlights e Saves time no need to re run gels e Saves precious sample re probe the mem brane using the same target sample e Provides efficient removal proprietary for mulation 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 with reducing agents e Economical less expensive than other com peting stripping buffers References Baolin Zhang 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 chemilumines cent 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 PRODUCT DESCRIPTION PKG SIZE 21059 Restore Western Blot 500 ml Stripping Buffer Sufficient for stripping 25 8 cm x 10 cm blots 21062 Restore Western Blot 30 ml Stripping Buffer Sufficient for stripping 1 8 cm x 10 cm blot Increasing the Sensitivity of a Western Blot Qentix ken tiks Western Blot Signal Enhancer It s like intensifying screens in a bottle There are many ways to increase the sensitivity of a Western blot Some method
47. blotting In the event that the protein is unable to re fold to create an intact binding site it may be necessary to add a denaturation renaturation step to the procedure or to perform the protein protein interaction in gel without transfer See In Gel Far Western Detection section that follows Denaturation renaturation is typical ly accomplished using guanidinium hydrochloride Blocking Buffer After transferring proteins to the membrane Western blotting procedures require that unre acted binding sites on the membrane be blocked with a non relevant protein solution In addition to blocking all remaining binding sites on the membrane a blocking buffer reduces nonspecific binding and aids in protein renaturation during the probing procedure A variety of different protein blockers may be used and no one blocking protein solution will work for all blotting experiments Any given protein blocker may cross react or otherwise disrupt the specific probing interaction being studied Determination of an effective blocking buffer must be made empirically Often bovine serum albumin BSA is used as a starting point for many membrane probing reactions Insufficient blocking may result in high background whereas prolonged blocking could result in a weak or masked signal Renaturation of the protein also appears to occur during the blocking step so it is important to optimize the blocking conditions to obtain the best signal to noise ratio for each applica
48. blotting procedure is to separate the macromolecules using gel electrophoresis Following electrophoresis the separated molecules are transferred or blotted onto a second matrix generally a nitrocellulose or polyvinylidene fluoride 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 or fluorogenic precipitate on the membrane for col orimetric or fluorometric detection respectively The most sensitive detection methods use a chemiluminescent 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 phosphorimager 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 membrane Detailed procedures for detection of a Western blot vary widely One common variation involves direct vs indirect detection as shown in Figure 1 With the direct detection method the primary antibody that is used to detect an antigen on the blot is also labeled with an enzyme or fluorescent dye This detection method is not widely used as most researchers pre fer the indirect detection method for a variety of
49. body 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 additional 5 15 minutes Some antigen antibody systems require an increase in temperature and or longer incubation periods Analysis of the successful removal of immunoprobes is recommended to prevent removal of the antigen or the unsuccessful removal of the antibodies After it has been determined that the membrane is free of immunodetection reagents a sec ond 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 reprob ings may result in a decrease in signal if the antigen is labile in Restore V 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 appli cations o Parom et Salsa Immunoassay lt H Strip Blot with Restore Western Blot Stripping Buffer N B Removal of Primary Antibody and HRP Conjugate A Removal of HRP Conjugate SuperSignal West Substrate i SuperSignal West Substrate Perform Immunoassay 2 gt Figure 26 Restore Western Blot Stripping Buffer Protocol Tel 800 874 3723 or 815 968 0747 www piercenet com Featured Product Restore
50. commend SuperSignal West Substrates over other chemiluminescent substrates for use in their instruments The high signal intensity and long signal duration make them ideal and sometimes essential for use in these instruments 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 blotting 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 accom plish 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 visi ble 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 it in Erase It9 Background Eliminator Solution Product 21065 which effec tively decreases the initial exposure time 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 i
51. commended starting dilution Costs are based on January 2004 U S list prices for an 8 x 10 cm mini gel following manufacturer s instructions Table 7 A comparison of SuperSignal West Substrates SuperSignal West Pico SuperSignal West Dura SuperSignal West Femto Chemiluminescent Substrate Extended Duration Substrate Maximum Sensitivity Substrate Primary Benefit e Twice the signal for about half Extended signal duration is e The most sensitive chemiluminescent the price of competing products ideal for use with imaging equipment substrate for HRP detection available Lower Detection Limit e Low picogram 10 12 e Mid femtogram 10 14 e Low femtogram 10 15 e Mid attomole 10 17 e High zeptomole 10 19 e Mid zeptomole 10 20 Signal Duration e 6 8 Hours e 24 Hours e 8 Hours Suggested Antibody Dilutions e Primary 1 1 000 1 5 000 e Primary 1 1 000 1 50 000 e Primary 1 5 000 1 100 000 e Secondary 1 20 000 1 100 000 Secondary 1 50 000 1 250 000 e Secondary 1 100 000 1 500 000 Room Temperature RT e 24 Hours e 24 Hours e 8 Hours Working Solution Stability Stock Solution Shelf Life e 1 year at RT 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 re
52. d 1 1 000 respectively Lanes 6 and 7 cor respond to yeast GFP lysate 1 10 and 1 100 respectively NOTE The UnBlot In Gel Chemiluminescent Detection Kit has been tested successfully with Novex FMC BioWhittaker and Bio Rad Criterion brand gels e The UnBlot In Gel Chemiluminescent Detection Kit does not perform well with Bio Rad Ready Gels Precise Protein Gels or Gradipore iGels 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 The recommended cross linking of gel is 8 18 4 20 and 10 20 gradient When using UnBlot Technology with homemade gels the glass plates must be siliconized prior to pouring the gel Please visit the Pierce web site to review the protocol and see other tips on optimizing UnBlot Technology o PRODUCT DESCRIPTION PKG SIZE 33500 UnBlot In Gel Chemiluminescent Kit Detection Kit Rabbit Sufficient reagents to perform 10 mini gel detections Includes UnBlot Substrate 110 ml Stabilized Goat anti Rabbit HRP 10 ul Dilution Buffer 50 ml BupH Pack PBS Buffer 17 packs Tween 20 5x10 ml Hands Off Incubation Colander 1 unit Pre cut Cellophane 10 sheets CL XPosure Film 5 x 7 25 sheets 33505 UnBlot 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 pl 33
53. d ELISA applications Starting Block Blocking Buffers are also avail able with an optimized amount of Tween 20 to provide the lowest background PKG PRODUCT DESCRIPTION SIZE 37539 StartingBlock T20 PBS 1 liter Blocking Buffer A protein based blocker formulation in phosphate buffered saline at pH 7 5 with 0 05 Tween 20 and Kathon Antimicrobial Agent 37543 StartingBlock T20 TBS 1 liter Blocking Buffer A protein based blocker formulation in Tris buffered saline at pH 7 5 with 0 05 Tween 20 and Kathon Antimicrobial Agent Featured Products 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 very high signal to noise ratio Plates blocked with SuperBlock Blocking Buffer can be stored dry for up to 12 months Liquid formulations available in PBS or TBS 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 packa
54. d 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 over a period of several hours allowing consistent and sensitive detection of proteins When the antibody is not diluted sufficiently too much enzyme is present and the substrate is used up quickly A stable output of light will never be achieved This is the single greatest cause of symptoms such as variability dark background with clear bands and decreased Sensitivity in Western blotting experiments with chemiluminescence To avoid this problem it is crucial to optimize the amount of antibody used for detection Antibody suppliers typi cally suggest a dilution range for using their antibody on a Western 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 o Table 5 Advantages of enhanced chemilumi nescence 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 Nonhazardous e No
55. d transferred overnight to nitrocellulose The membrane was blocked with SuperBlock Blocking Buffer in PBS Product 3 515 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 Product 34075 was added The blot was exposed to film for 10 seconds and resulted in a completely black image A Using the old option to resolve the problem of a completely dark film 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 Product 34075 The blot was exposed to film for 10 seconds This optimization required a two day procedure B Using the new option the initial dark film A was treated with Erase It Background Eliminator to allow the band images to appear in 4 minutes C For more product information or to download a product instruction booklet visit www piercenet com Highlights e Reduces signal evenly over the film no altering of results e Fast easy background elimination from over exposed 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 obtai
56. d yeast GFP extract were separated by SDS PAGE One gel was transferred to nitrocellulose membrane After transfer the mem brane 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 UnBlot In Gel Chemiluminescent Detection Kit Rabbit Product 33500 Signal was detected using UnBlot Substrate The lanes on the gel and in the membrane are as follows Lanes 1 2 and 3 cor respond 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 23 4 5 23 4 5 6 7 Figure 25 Versatility and specificity of the UnBlot System E coli bacterial GFP 6xHis tagged lysate Pure GFP 6xHis tagged and yeast GFP extract were separated by SDS PAGE Both gels were pre treated with 50 isopropanol The antigens were detected using a 1 1 000 dilution of GFP Monoclonal Mouse Gel 1 or of a 1 500 dilution of anti Penta his mouse antibody Gel 2 and the UnBlot In Gel Chemiluminescent Detection Kit Rabbit Product 33500 Signal was detected using UnBlot Substrate 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 an
57. denaturing systems may not ampules always present an interface that promotes the intended interaction UnBlot Stable Peroxide 22m UnBlot Luminol Enhancer 55 ml e Reduced nonspecific binding biotin streptavidin HRP systems demonstrate less E D a ee TH A A f aa E roroun ar Western mini gels nonspecific binding compared to antibodies directed against the bait protein the anti GST Protein Protein Interaction Kit antibody conjugate is highly specific for the GST tag Materials and methods for the discovery in gel or on membrane of protein e Compatible with protein staining can be used for total protein staining after the chemi mo GST tagged bait n protein as the prove luminescent detection step eliminating the need to run two gels includes Anti Glutathione 0 25 mg S Transferase GST HRP Dilution Buffer 10X 90 ml BupH Phosphate Buffered 17 packs Saline 10 Tween 20 6 x 10 ml ampules UnBlot Stable Peroxide 55 ml UnBlot Luminol Enhancer 55 ml Cellophane Exposure Sheets 10 pack Tel 800 874 3723 or 815 968 0747 www piercenet com In Gel Western Detection 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 invol
58. dmondson D G and Dent S Y R 2001 Current protein to renature The gel is then incubated with the bait protein The bait protein is then detect Protocols in Protein Science 19 7 1 19 7 10 ed with an HRP tagged antibody or biotin binding protein Golemis E Ed 2002 Protein Protein Interactions u IEEE A Laboratory Manual Cold Spring Harbor Laboratory The same controls and experimental conditions necessary for optimization of membrane based Press Product 20068 far Westerns apply to in gel detection With in gel detection the blocking step can be eliminated Kaelin W G et al 1992 Cell 70 351 364 but the bait protein and the labeled detection protein must be diluted in the blocking buffer to Reddy V M and Kumar B 2000 J Infect Dis 181 reduce nonspecific binding Also higher amounts of prey and bait proteins are often required for 1189 1193 detection compared to membrane detection with the equivalent chemiluminescent substrate ProFound Far Western Protein Protein Interaction Kits Pierce provides two kits for far Western analysis These kits are optimized for detection both on membrane or in gel One kit allows the detection of biotinylated bait proteins Product 23500 and the other allows for the detection of GST tagged bait proteins Product 23505 Both kits include blocking and wash buffers HRP labeled detection protein Streptavidin HRP or Anti GST HRP and an extremely sensitive formulati
59. e Stabilizer Diluent SD 1 liter 31503 SuperFreeze Peroxidase Conjugate Stabilizer 25 ml 29810 Ethylene Glycol 200 ml 50 aqueous solution For more product information or to download a product instruction booklet visit www piercenet com 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 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 laborato ry Chapters have been entirely rewritten reorganized and updated to provide background context and step by step instructions for tech niques 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 pro teins and epitope mapping Rather than presenting an array of solutions for working with antibodies and antigens Using Antibodies identifies the best approach to specif ic problems These recommendations include more detail in the protocols extensive advice on avoiding and solving problems information regarding proper controls and thorough illustra tion of theory methods and results The book also includes a bonus a
60. e a secondary anti body 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 proteins includ ing antibodies from those species To expedite the process of choosing the appropriate secondary antibody visit the Secondary Antibody Selection Guide on the Pierce web site located under the Products tab or the home page Antibodies for Western blotting are typically used as dilute solutions ranging from 1 100 1 500 000 dilutions 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 detection systems require that less antibody be used which can result in substantial savings on antibody costs and allow a limited supply of antibody to be stretched out over more experiments It also produces a side benefit of reduced background because the limited amount of antibody shows increased specificity for the target with the highest affinity Antibody dilutions are typically 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 Pierce offers a wide variety of ImmunoPure Labeled Secondary Antibodies for use in Western blotting The labels include biotin fluorescein rhodamine horseradish peroxidase and alkaline phosphatase
61. e optimal results Longer exposure times may be necessary as the blot ages Tel 800 874 3723 or 815 968 0747 www piercenet com Recommended Readine Bers G and Garfin D 1985 Protein and nucleic acid blotting and immunobiochemical detection BioTechniques 3 276 288 Bjerrum O J and Heegaard N H H 1988 Handbook of Immunoblotting of Proteins Volume 1 Technical Descriptions CRC Press Boca Raton Bollag D M et al 1996 Protein Methods Second Edition Wiley Liss Inc New York Product 20001 Gallagher S 1996 Immunoblot Detection Current Protocols in Protein Science pp 10 10 1 10 10 11 John Wiley and Sons Inc New York Gershoni J 1988 Protein blotting 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 Ca
62. ectly from proteins Horseradish Peroxidase peptides or other ligands that contain a free SH group Two reagents SATA and mercap 31494 Sisi usos ipe Kit toethylaminesHCl are also included in the kit formats to produce free sulfhydryls on Fade n ane macromolecules for conjugation Activated Horseradish Peroxidase Activated Horseradish Peroxidase 20 ml EZ Link Maleimide Activated Peroxidase References Conjugation Buffer 2 MercaptoethylamineeHCI 6 mg Choi J Y et al 2002 J Biol Chem 277 21630 21638 SATA 2 mg Seo Y R et al 2002 PNAS 99 14548 14553 Dimethylformamide 1ml HydroxylamineeHCl 5 mg Yoo J H et al 2004 J Biol Chem 219 048 859 Polyacrylamide Desalting Column 1x10 ml For more product information or to download a product instruction booklet visit www piercenet com Labeling Your Own Antibodies aos Qe um 29 Enzyme containing Enzyme with polysaccharide chains reactive aldehyde groups NaCNBH NH 0 0 WY N B 0 Reductive amination forms stable secondary amine linkage Antibody molecule containing amine groups Figure 9 Conjugation scheme for periodate oxidation and subsequent reductive amination Periodate Glycoproteins such as horseradish peroxidase glucose oxidase and most anti body molecules can be activated for conjugation by treatment with periodate Oxidizing polysaccharide residues in a glycoprotein with sodium periodate provides a mild and efficient way of gen
63. ein detection application A novel treatment reactive chemical blocking RCB may be used to eliminate this carboxyl binding motif thus impart ing exclusive specificity toward phosphate groups PhosphoProbe HRP in conjunction with RCB is a universal phosphate detection probe PhosphoProbe HRP has been opti mized for direct detection of phosphoester molecules such as nucleotides or protein peptides containing phosphoserine phosphothreonine and phosphotyrosine Carboxyls are selectively converted to amines 0 PO COOH EDC amp EDA PO C N C C NH FE3 Phospho N gt Phospho protein Reactive chemical protein Addition of blocking step PhosphoProbe HRP COOH PO 0 PO NHy C C N C H o Comparison of Polyhistidine tagged PHT Fusion Protein Detection Methods A B Figure 21 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 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 10 x 500 ml Saline Packs Surfact Amps 20 10 6 x 10 ml ampules 15166 PhosphoProbe HRP 2 mg 23031 Ethylenediamine Dihydrochloride 10g 22980 EDC 5 22981 EDC 250 15167 PhosphoProbe Phosphorylated Kit Protein Detecti
64. ends of glutaraldehyde will couple with amines to form secondary amine linkages The reagent is highly efficient at protein conju gation but has a tendency to form various high molecular weight polymers making results difficult to reproduce EZ Link Activated Peroxidase References Sandt C H and Hill C W 2001 nfect Immun 69 7293 7303 Turpin E A et al 2003 J Clin Microbiol 41 3579 3583 EZ Link Plus Activated Peroxidase References Glover L 2002 Eur J Biochem 269 4607 4616 Nawa M et al 2000 Clin Diagn Lab Immunol 7 114 111 V lkel T et al 2001 Protein Eng 14 815 823 PRODUCT DESCRIPTION PKG SIZE 31487 EZ Link Plus Activated 1 mg Peroxidase Periodate Activated 31488 EZ Link Plus Activated 5x1mg Peroxidase Periodate Activated 31489 EZ Link Plus Activated Kit Peroxidase Kit Periodate Activated Includes EZ Link Plus 5x1mg Activated Peroxidase Sodium Cyanoborohydride 1x0 5ml Solution Quenching Buffer 25 ml BupH Phosphate Buffered 500 ml Saline Pack BupH Carbonate Buffer Pack 500 ml 31496 EZ Link Activated Peroxidase 1 mg Glutaraldehyde Activated 31495 EZ Link Activated Peroxidase 5 mg Glutaraldehyde Activated 31497 EZ Link Activated Peroxidase Kit Antibody Labeling Kit Glutaraldehyde Activated Includes EZ Link Activated 5 mg Peroxidase Conjugation Buffer 50 ml Lysine 250 mg Immobilized Protein A G Column 0 5 ml Gent
65. ensitometry data on dot blot comparing before and after use of the Erase It 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 Erase It8 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 results showed that signal intensity decreased evenly with time when treated with the Erase It Solution maintaining similar slopes on a dose response curve o Erases Speckling Before using After using Erase It8 Erase It Solution Solution A B Figure 35 Recombinant Human TNFa was electro phoresed on a 4 20 SDS polyacrylamide gel and transferred to a nitrocellulose membrane The mem brane was blocked and detected with mouse anti human TNFa followed by goat anti mouse HRP Product 7 31434 and SuperSignal West Dura Substrate Product 34075 The blot was exposed t
66. erSignal West Dura Substrate provides the maximum light duration which allows for multiple extended exposures an IL 2 blot was repeated in comparison to the acridan based HRP chemiluminescent substrate ECL Plus Substrate using the manufactur er s recommended dilutions and membrane ECL Plus Substrate strongly recommends the use of PVDF The proteins were transferred to PVDF for the ECL Plus Substrate and to nitro cellulose for the SuperSignal System The primary antibody for both substrates was used at a 1 ug ml dilution A 10 ng ml dilution was used for the secondary antibody SuperSignal West Dura Substrate and a 20 ng ml dilution was used for ECL Plus Substrate A five minute film exposure showed a high signal to noise ratio for the SuperSignal West Dura System with detection down to 3 pg of IL 2 Figure 17A It showed high background for the acridan system and detection down to only 800 pg of IL 2 Figure 17B A 30 minute exposure at F1 6 on the CCD camera demonstrated detection down to 12 5 pg of IL 2 with the SuperSignal9 Product Figure 17C When the ECL Plus Blot was exposed to the CCD camera at F1 6 the expo sure was stopped at 15 minutes because of the intense background Signal was difficult to distinguish above background Figure 17D Reference Tokumaru H et al 2001 Cell 104 421 432 Supersignal West Dura Substrate 9 minutes film 90 2512 56 3 3 1 1 6 0 8 0 4 0 2 0 1 05 03 013 006 003 ng
67. erating reactive aldehyde groups for subsequent conjugation with amine or hydrazide containing molecules via reductive amination Figure 9 Some selectivity of mono saccharide oxidation may be accomplished by regulating the concentration of periodate in the reaction medium In the presence of 1 mM sodium periodate sialic acid groups are specifical ly oxidized at adjacent hydroxyl residues cleaving off two molecules of formaldehyde and leaving one aldehyde group At higher concentrations of sodium periodate 10 mM or greater other sugar residues will be oxidized at points where adjacent carbon atoms contain hydroxyl groups This reaction should be performed in the dark to prevent periodate break down and for a limited period of time 15 30 minutes to avoid loss of enzymatic activity Cross linking with an amine containing protein takes place under alkaline pH conditions through the formation of Schiff base intermediates These relatively labile intermediates can be stabilized by reduction to a secondary amine linkage with sodium cyanoborohydride Reductive amination has been done using sodium borohydride or sodium cyanoborohydride however cyanoborohydride is the better choice because it is more specific for reducing Schiff bases and will not reduce aldehydes Small blocking agents such as lysine glycine ethanolamine or Tris can be added after conjugation to quench any unreacted aldehyde sites Ethanolamine and Tris are the best choices for block
68. ere removed and placed in sheet protectors prior to exposure to film for 30 seconds and 5 minutes as indicated The film was developed per the manufacturer s instruc tions The resulting blots were analyzed for signal noise and compared The results indicate that there is no blocking reagent that is optimal for all systems Tel 800 874 3723 or 815 968 0747 www piercenet com Featured Product StartingBlock Blocking Buffer Confused about all the blocking options available for Western blot and ELISA applications StartingBlock Blocking Buffer simplifies the selection of a blocker Although no blocking buffer is ideal for every system you can improve the odds dramatical ly with StartingBlock Blocking Buffer because it is compatible with the widest variety of antibodies For example StartingBlock Blocking Buffers are compatible with biotin containing sys tems while milk based protein blockers interfere StartingBlock Buffers do not cross react with rabbit antibodies while many other blockers do StartingBlock Blocking Buffers are also free of potentially interfering serum proteins StartingBlock Blocking Buffers offer a high level of performance regardless of the sys tem you choose for your Western blotting or ELISA application In fact they may be the only blockers you ever use 7A 7B Figure 7A 7B IC ID Membrane Type Nitrocellulose PVDF Nitrocellulose PVDF Film Exposure Time 30
69. es 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 imag ing instruments The instruments have their own light proof boxes e Refer to the instrument manufacturer s instructions for more information on an indi vidual instrument Highlights e Up to one third the price of competitive prod ucts e Provides the same detection sensitivity as other commercially available films e Available in either 5 x 7 or 8 x 10 sheets in packages of 25 50 or 100 non interleaved sheets Reference Tikhonov I et al 2003 J Virol TT 3157 3166 PRODUCT DESCRIPTION PKG SIZE 34090 CL XPosure Film 100 sheets 5 x 7 sheets 34091 CL XPosure Film 100 sheets 8 x 10 sheets 34092 CL XPosure Film 25 sheets 5 x 7 sheets 34093 CL XPosure Film 50 sheets 8 x 10 sheets Featured Products SuperSignal West Pico HisProbe Kit Specific 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 polyhistidine tags reducing background problems Unlike anti His antibodies HisProbe HRP can recognize
70. f 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 signal 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 detection is the ability to strip reagents from a blot and then reprobe the same blot This is possible with chemilumines cence because all of the reagents can be removed from the membrane 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 causing a significant amount of antigen to be released from the membrane Various proto cols have been proposed to accomplish this purpose and they generally include some combination of detergent reducing agent heat and or low pH During the stripping proce dure some amount of antigen is inevitably lost from the membrane It is important to minimize this loss by st
71. fers tools for a variety of antibody modification strategies Understanding the functional groups available on an antibody is the key to choosing the prop er method for modification For example Primary amines NH are found on lysine residues and the N terminus These are abun dant and distributed over the entire antibody Sulfhydryl groups SH are found on cysteine residues and are formed by selectively reducing disulfide bonds in the hinge region of the anti body Carbohydrate residues containing cis diols can be oxidized CHO to create active aldehydes These are localized to the Fc region on antibod ies and are more abundant on polyclonal antibodies Labeling Your Own Antibodies Horseradish Peroxidase lts high specific enzyme activity makes it the enzyme of choice Highlights e Superior to alkaline phosphatase and B galactosidase conjugates due to the higher specif ic enzyme activity e Small size 40 kDa allows excellent cellular penetration e Variety of substrates available e deal in blotting and cytochemistry applications e Used as the reporter enzyme for SuperSignal Chemiluminescent Substrates References Cordell J L et al 1984 J Histochem Cytochem 32 219 229 Hosoda H et al 1987 Chem Pharm Bull 35 3336 3342 Passey R B et al 1977 Clin Chem 23 1 131 139 Porstmann B et al 1985 J Immunol Methods 79 27 37 Samoszuk M K et al 1989 Antibody Im
72. ging 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 T 942 951 Subbarayan V et al 2001 Cancer Res 61 2720 276 Walters R W et al 2002 Cell 100 789 799 PRODUCT DESCRIPTION PKG SIZE 37515 SuperBlock PBS Blocking Buffer 1 liter 37516 SuperBlock T20 PBS Blocking Buffer Contains 0 05 Tween 20 1 liter 37535 SuperBlock TBS Blocking Buffer 1 liter 37536 SuperBlock T20 TBS Blocking Buffer Contains 0 05 Tween 20 1 liter 37517 SuperBlock PBS Blocking Buffer Blotting 1 liter 37537 SuperBlock TBS Blocking Buffer Blotting 1 liter 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 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 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 PRODUCT DESCRIPTION PKG SIZE 37527 SEA BLOCK Blocking Buffer 500 ml Blocker Casein Read
73. hat was found in the polyacrylamide gel Gel Transfer Membrane Electrophoretic Transfer Filter Paper Gel Membrane Filter Sandwich Buffer Tank Electrodes cathode Direction of Transfer Figure 2 Electrophoretic transfer Transfer efficiency can vary dramatically among proteins based upon the ability of a protein to migrate out of the gel and its propensity to bind to the membrane under a particular set of conditions The efficiency of transfer depends on factors such as the composition 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 pres ence of detergents Optimal transfer of proteins is generally obtained in low ionic strength buffers and with low electrical current Pierce offers a wide selection of the most commonly used membranes for Western blotting including nitrocellulose and polyvinylidene difluoride PVDF Please refer to page 6 for a complete offering of transfer membranes At this stage before proceeding with the Western blot it is often desirable to stain all pro teins on the membrane with a reversible stain to check the transfer efficiency Although the gel may be stained to determine that protein left the gel this does not ensure efficient bind ing of protein on the membrane Ponceau S stain is the most widely used reagent for staining proteins on a
74. health hazards e No waste disposal problems Stable e Unlike radioisotopes the shelf life is long e Store at 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 remains glowing for an extended peri od of time e Ability to place blot against film at various times e Ability to optimize the developing method Ability to reprobe the blot e Nonisotopic probes can be stripped off the membrane e mmunodetection can be repeated Large linear response e Can detect a large range of protein concentra tions Quantitative e The X ray film can be scanned using a reflectance densitometer or using an imaging device such as a CCD camera Tel 800 874 3723 or 815 968 0747 www piercenet com Chemiluminescent Substrates Table 6 Blotting cost comparison between SuperSignal ECL and Western Lightning Products SuperSignal Western West Pico ECL Lightning Substrate Cost Comparison Substrate Substrate Substrate Membrane 8 x 10 or 8 x 12 5 66 8 30 12 40 TBS Wash Buffer 1 24 1 24 1 30 SuperBlock Blocking Buffer 4 48 4 48 4 69 Primary Antibody 3 15 31 50 5 78 Secondary Antibody 0 04 0 47 0 47 Substrate 3 72 5 46 7 88 Film 1 20 2 28 2 12 Total Blotting Cost 19 49 53 73 34 64 Endogen s Anti CD54 Product MA5407 500 ug was used at the substrate manufacturer s re
75. ields a brown precipitate in the presence of HRP and per oxide 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 pro duced by the metal complexes is darker than the color produced by DAB alone enhancing the sen sitivity in staining applications PRODUCT DESCRIPTION PKG SIZE 34002 DAB Substrate Kit 275 ml Includes DAB 10X Stable 25 ml Peroxide Buffer 250 ml 34065 Metal Enhanced DAB Substrate Kit 275 ml 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 pre cipitate that photographs well The CN DAB Substrate works well in Western blotting and dot blotting applications PRODUCT DESCRIPTION PKG SIZE 34000 CN DAB Substrate Kit 275 ml Includes CN DAB 10X 25 ml Stable Peroxide Buffer 250 ml Tel 800 874 3723 or 815 968 0747 www piercenet com Chromogenic Substrates Substrates for Alkaline Phosphatase NBT with a molecular weight of 817 6 is a member of a class of heterocyclic organic com pounds known as tetrazolium salts Upon reduction the compound yields NBT formazan a highly colored
76. iliz er Table 4 lists the typical working dilutions for the conjugated antibodies when doing ELISAs immunoblotting or immunohistochemical techniques Table 4 Typical dilution ranges recommended for Pierce ImmunoPure Polyclonal Conjugated Antibodies Conjugate ELISA Immunoblotting Immunohistochemistry Alkaline 1 5 000 1 50 000 1 2 500 1 25 000 1 500 1 5 000 Phosphatase Peroxidase 1 5 000 1 200 000 1 25 000 1 500 000 1 500 1 5 000 for SuperSignal9 for SuperSignal ELISA Products West Products Fluorescein 1 50 1 200 Rhodamine 1 50 1 200 Storing Enzyme Conjugates Pierce provides a variety of reagents to help preserve enzyme conjugate 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 SuperFreeze Peroxidase Conjugate Stabilizer Product 31503 diluting the conjugate 1 1 in the stabilizer and storing it at 20 C for up to one year as a stock solution Guardian Peroxidase Stabilizer Diluents Product s 37548 and 37552 allow peroxidase conjugates to be reconstituted and stored at 4 C as a 1 1 000 dilution or a 1 100 000 dilution stock solution Conjugate Stabilizers PRODUCT DESCRIPTION PKG SIZE 37548 Guardian Peroxidase Conjugate Stabilizer Diluent SD 200 ml 37592 Guardian Peroxidase Conjugat
77. iment to another e Little signal amplification Advantages of Indirect Detection Figure 1B e Sensitivity is increased because each pri mary antibody contains several epitopes that can be bound by the labeled second ary antibody allowing for signal amplification e A wide variety of labeled secondary anti bodies are available commercially e Since many primary antibodies can be made in one species and the same labeled Secondary antibody can be used for detec tion it is versatile e Immunoreactivity of the primary antibody is not affected by labeling e Different visualization markers can be used with the same primary antibody Disadvantages of Indirect Detection Figure 1B e Cross reactivity may occur with the sec ondary antibody resulting in nonspecific Staining e An extra incubation step is required in the procedure Tel 800 874 3723 or 815 968 0747 www piercenet com SDS PAGE Separate protein sample by electrophoresis e PAGEprep Protein Clean up and Enrichment Kit Product 26800 Precise Protein Gels many available see www piercenet com e Tris HEPES SDS Running Buffer Product 28398 e ImmunoPure Lane Marker Reducing Sample Buffer 5X Product 39000 e mmunoPure Lane Marker Non Reducing Sample Buffer 5X Product 39001 e BlueRanger Prestained Protein Molecular Weight Marker Mix Product zs 26681 and 26685 e Chemiluminescent BlueRanger Prestained Peroxidase l
78. imum Sensitivity Substrate provides the ultimate sensitivity for Western blotting allowing users to see protein bands that were never visualized before Highlights e Sensitive reach low femtogram detection limits that s zeptomole level detection e Economical conserve precious primary antibodies with up to 1 100 000 dilutions and secondary antibodies to 1 500 000 e Comes with HRP labeled secondary antibodies e Intense releases the most intense signal generated by chemiluminescent systems mak ing it easy to capture an image on film or via an imager system Lower detection limit e Low femtogram 1075 e Mid zeptomole 10 20 Signal duration e 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 4C or 6 months at RT Supersignal West Acridan based Femto Substrate Substrate Fee EcL Pres j i S amp gt b Nitrocellulose Nitrocellulose Figure 18 A comparison between SuperSignal West Femto and ECL Plus Substrates Two fold serial dilutions of mouse IL 2 from 1 000 to 15 6 pg were detected with both substrates The primary antibody dilution was 1 2 000 and the secondary antibody dilution was 1 300 000 Both antibodies had a 1 mg ml starting concentration For more product information or to download a product instruction booklet visit www piercenet com o PRODUCT DESCRIPTION PKG SIZE 34
79. in and then incubated with sub strate that was prepared according to the manufacturer s instructions Blots were exposed to film for one and five minute exposures Tel 800 874 3723 or 815 968 0747 www piercenet com Featured Product Table 8 A conversion protocol for using SuperSignal Substrates Step by step Conversion Protocol 1 Perform standard electro phoresis and blotting 2 Block the nonspecific sites 3 Add diluted primary antibody incubate for 1 hour then wash 4 Add diluted secondary antibody HRP labeled incubate for 1 hour then wash 9 Prepare chemiluminescent substrate 6 Incubate the substrate on the blot 7 Expose to film References ECL Substrate Use their Hybond Nitrocellulose Membrane Add blocking reagent incubate and wash Optimization Range 1 100 1 1 500 dilution Optimization Range 1 1 500 1 50 000 dilution Mix equal volumes of both solutions Incubate blot with Working solution without agitation for precisely 1 minute It s recommended that you work quickly once ECL Working Solution has been added to the membrane Immediately expose to film for 1 minute 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 For more product information or to download a product instruction booklet visit www piercenet com
80. ing agents because they contain hydrophilic hydroxyl groups with no charged functional groups The pH of the reductive amination reaction can be controlled to affect the efficiency of the cross linking process and the size of the resultant antibody enzyme complexes formed At physiological pH the initial Schiff base formation is less efficient and conjugates of lower molecular weight result At more alkaline pH i e pH 9 10 Schiff base formation occurs rap idly and with high efficiency resulting in conjugates of higher molecular weight and greater incorporation of enzyme when oxidized enzyme is reacted in excess Low molecular weight conjugates may be more optimal for immunohistochemical staining or blotting techniques in which penetration of the complex through membrane barriers is an important consideration Washing steps also more effectively remove excess reagent if the conjugate is of low molecu lar weight thus maintaining low background in an assay By contrast conjugates of high molecular weight are more appropriate for ELISA procedures in a microplate format where high sensitivity is important and washing off excess conjugate is not a problem Glutaraldehyde Another method for conjuga tion uses glutaraldehyde one of the oldest homo bifunctional cross linking reagents used for protein conjugation It reacts with amine groups to create cross links by one of several routes Under reducing conditions the aldehy des on both
81. inol is oxidized in the presence of horseradish peroxidase 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 throughout the past decade because they offer several advantages over other detection methods These advantages have allowed chemiluminescence to become the detection method of choice in most protein labo ratories Using chemiluminescence allows multiple exposures to be performed to obtain the best image The detection reagents can be stripped away 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 over 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 Femto Chemiluminescent substrate Product 34095 lower detection limits in the low femtogram range are possi ble 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 colore
82. ion Kit 34082 SuperSignal West Pico Kit Mouse IgG Detection Kit 34084 SuperSignal West Pico Complete Kit Rabbit IgG Detection Kit 34083 SuperSignal West Pico Kit Rabbit IgG Detection Kit 34086 SuperSignal West Pico Complete Kit Biotinylated Protein Detection Kit 34085 SuperSignal West Pico Kit Biotinylated Protein Detection Kit Featured Product SuperSignal West Dura Extended Duration Substrate Specially formulated for use with CCD cameras Along with advances in cooled CCD technology has come an overall reduction in the cost of imaging instruments It is increasingly common to see these instruments in today s research labs Pierce developed SuperSignal West Dura Extended Duration Substrate to meet the needs of researchers using this efficient new technology Cooled CCD cameras which offer the advantages of instant image manipulation higher sensitivity greater resolution and a larger dynamic range than film also eliminate the need for film processing equipment and a darkroom However this technology has one drawback it requires a substrate that pro duces an intense signal that is strong enough and of long enough duration to be captured by the cameras Pierce developed SuperSignal West Dura Substrate to meet this need 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 Because Sup
83. l Kits Step 1 Preparation of Protein Step 2 Labeling Reaction For salt free lyophilized protein Step 3 Removal of Excess Fluorescent Dye Dissolve in borate buffer NN he For sample volume of 100 ul or less Exchange into PBS buffer using AN W Slide A Lyzer MINI Dialysis Unit EN 7 fe 7 For proteins in buffers or salt solutions c a Sample volume of 100 ul or less Exchange into borate buffer using Slide A Lyzer MINI Dialysis Unit Peconi NU Www For sample volume greater than 100 ul ee MSN Add dye to the Exchange into PBS buffer using a dye with DMF protein solution D Salt Dextran Column ww b Sample volume greater than 100 ul 4 Incubate for 1 hour Exchange into borate buffer using a D Salt Dextran Column For more product information or to download a product instruction booklet visit www piercenet com Optimizing Antibody Concentration uz iz I 12 2 12 1 12 1 1 55 I2 14 L8 Eee Bex 1 2 ta 4 Mouse anti Human p53 1 500 1 500 1 1 000 1 1 000 1 ug ml 1 ug ml 0 5 ug ml 0 5 ug ml Goat anti Mouse HRP 1 1 000 1 5 000 1 10 000 1 20 000 1 ug ml 0 2 ug ml 0 1 ug ml 0 05 ug ml Exposure Time 30 seconds 30 seconds 1 minute 1 minute Figure 10 Example of signal intensity on a Western blot when using SuperSignal West Pico Substrate and antibodies at various concentrations Recombinant Human Wild Type p53 Baculovirus lysate at various concentrations
84. ld not interact nonspecifi cally with the bait protein In approaches that use a secondary system for detection of the prey protein such as enzyme labeled streptavidin with a biotinylated bait protein it is important to include a duplicate control membrane that is probed only with the labeled streptavidin This would reveal any bands resulting from endogenous biotin in the Sample or nonspecific binding of the labeled Streptavidin When a fusion tag is used with a corresponding antibody it is critical to probe one of the control membranes with the labeled antibody alone This control helps to confirm that the relevant band is not due to nonspecific binding of the labeled secondary antibody To obtain meaningful results appropriate test and control experiments should be subjected to gel electrophoresis transfer and probing in parallel In Gel Far Western Detection Advantages of In Gel Detection References Blackwood E M and Eisenman R N 1991 Science Because of restrictions associated with the transfer process blocking and the possibility of non 251 1211 1217 specific binding of bait proteins to unrelated bands on the membranes it is sometimes Burgress R R et al 2000 Methods Enzymol 328 advantageous to perform far Western detection within the gel In this procedure the gels are pre 141 157 treated with 50 isopropyl alcohol and water to remove SDS from the gel and to allow the prey E
85. le Ag Ab Binding Buffer 200 ml Gentle Ag Ab Elution Buffer 200 ml Tel 800 874 3723 or 815 968 0747 www piercenet com Featured Product EZ Label Fluorescent Labeling Kits PRODUCT DESCRIPTION PKG SIZE Make your own fluorescent labeled antibody in less than two hours 22009 bn ae i Ki Sufficient for five coupling reactions EZ Label Kits are designed for labeling any size protein small or large even if you have includes No Weight d 6x1mg only a small amount of your protein Protein sample volumes ranging from 50 ul 1 ml can Pre Measured Fluorescein microtubes be used with protein concentration up to 10 mg ml for each reaction EZ Label Kits were Dern mi Inm specially developed and optimized for the most efficient labeling BupH Borate Buffer Packs 5 packs BupH Phosphate Buffered 5 packs EZ Label Kits contain everything you need to successfully label your antibody or protein Saline Packs D Salt Dextran Desalting 5 columns e Fluorescent dye provided in individual microtubes eliminating the need to weigh dye Columns a Slide A Lyzer MINI Dialysis 5 units e Conveniently packaged dimethylformamide DMF to prepare the fluorescent dye solution Unit Pack e Pre made borate and phosphate buffers just add water to the powder and they are Reaction Tubes 5 tubes ready to use 93002 EZ Label Rhodamine Protein Kit e Pre packed ready to use desalting columns for fast buffer exchange when your protein Labe
86. ling NIE Sufficient for five coupling reactions sample volume is greater than 100 ul Includes No Weigh 6x0 5 mg e Slide A Lyzer MINI Dialysis Units for easy buffer exchange when your protein sample ee Rhodamine microtubes Icrotupes volume is less than or equal to 100 ul Dimethylformamide DME 1 mi e Amber reaction tubes no handling in the dark required BupH Borate Buffer Packs 5 packs BupH Phosphate Buffered 5 packs Excitati Emissi Saline Packs i xcitation mission D Salt Dextran Desalting 5 columns EZ Label Kit Wavelength nm Wavelength nm Columns Slide A Lyzer MINI Dialysis 5 units Fluorescein Protein Labeling Kit 491 518 Unit Pack Reaction Tubes 5 tubes Rhodamine Protein Labeling Kit 044 0 6 33004 EZ Label Fluorescein Kit lsothiocyanate FITC Fluorescein Isothiocyanate 494 520 Protein Labeling Kit Sufficient for five coupling reactions FITC Protein Labeling Kit Includes No Weigh 6x1 mg i Pre Measured microtubes These kits contain sufficient reagents to perform five fluorescent labeling reactions which use up to 10 FITC Microtubes mg ml of protein for each reaction 50 ul 1 ml volume of protein Dimethylformamide DMF 1 ml BupH Borate Buffer Packs 5 packs BupH Phosphate Buffered 5 packs Saline Packs D Salt Dextran Desalting 5 columns Columns Slide A Lyzer MINI Dialysis 5 units Unit Pack Reaction Tubes 5 tubes See how easy the fluorescent labeling procedure is when you use EZ Labe
87. lution A large number of nonspecific bands are also visible An opti mal blot was achieved by using a 1 5 000 primary antibody dilution and a 1 50 000 secondary antibody dilution o 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 find out which anti body concentration is most appropriate to that particular combination of membranes proteins and antibodies Optimization is even more cru cial when key components of a system are changed such as switching from a colorimetric substrate like chlororonaphthol CN to more sensitive chemiluminescent substrates such as SuperSignal West Products The first step of optimizing the blotting conditions usually involves optimizing the antibody concentrations or dilutions through the use of a dot blot pro tocol The next step is usually the optimization of the blocking buffer by testing cross reactivity of several different buffers with the blotting sys tem s key components see page 7 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 anti body will vary The optimal antigen and antibody concentrations can be determined by perf
88. lutions 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 Place dry nitrocellulose 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 Block the nonspecific sites on the nitrocellulose membranes by incubating them in blocking buffer that contains 0 05 Tween 20 blocker Tween 20 for 1 hour at RT with shaking Prepare the primary antibody dilutions 1 1 000 1 5 000 in blocker Tween 20 and apply to the membranes Incubate for 1 hour at RT with shaking SuperSignal SuperSignal SuperSignal Lumi Phos West Pico West Femto West Dura WB Substrate Substrate Substrate Substrate 1 1 000 1 5 000 or 0 2 1 0 ug ml 1 5 000 1 100 000 1 1 000 1 50 000 1 200 1 2 000 or 0 01 0 2 ug ml or 0 02 1 0 pg ml or 0 5 5 0 ug ml from 1 mg ml stock 6 Wash the membrane 4 6 times in TBS or PBS using as large a volume of wash buffer as possible Add 0 05 Tween 20 to the wash buffer to help reduce nonspecific back ground For each wash suspend the membrane in wash buffer and agitate for approximately 5 minu
89. membrane However it has limited sensitivity does not photograph well and fades with time Pierce MemCode Reversible Stain is a superior alternative for staining protein on nitrocellulose Product 24580 or PVDF Product 24585 mem branes MemCode 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 Szacmunelbzmse haallat RcR FUAF Bemeomsmenmlb cam instruction booklet VISIT www piercener com ae a a a nee 1 xe m o E sli ans cau mancare For more product information or to download a product o Featured Product MemCode Reversible Protein Stain for Nitro cellulose and PVDF A great NEW 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 MemCode Reversible Protein Stains decrease staining time increase staining sensitivity and enhance the immunoreactivity of antigens in subsequent Western blotting Try these new 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 to band erasure in minutes e Turquoise bands are easily photographed e Stained bands do not fade with time
90. minescent 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 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 5 iB Figure 28 Re probing with different antibodies Western blots of HeLa cell lysate protein diluted from 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 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 tmctnusctlan Laallat oeat RAF larcanatl cam instruction booklet VISIT www piercener com EL menm 2e mum sl dh Gueff m mnen subo enze 3 a pu anima sn semmoslunol For more product information or to download a product Fi r
91. minutes 30 minutes 24 hours 24 hours Full duration of SuperSignal West Dura Chemiluminescent Substrate light emission Figure 7A D Comparison of StartingBlock Blocking Buffer Performance after stripping and reprobing Nitrocellulose vs PVDF when probed for the transferrin receptor CD71 Highlights Compatible with a wide range of detection systems e Works in both Western and ELISA applications Does not cross react 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 noise ratios in the range of 10 1 20 1 have been realized with StartingBlock Blocking Buffer For more product information or to download a product instruction booklet visit www piercenet com PKG PRODUCT DESCRIPTION SIZE 37538 StartingBlock PBS 1 liter Blocking Buffer A protein based blocker formulation in phosphate buffered saline pH 7 5 for use in Western blotting and ELISA applications 37542 StartingBlock TBS 1 liter Blocking Buffer A protein based blocker formulation in Tris buffered saline pH 7 5 for use in Western blotting an
92. molecules mild reduction with Mercaptoethyl aes e M ena sale ymes Jellnig y uu ptoetyi Alkaline Phosphatase amineeHCI MEA results in two half antibody fragments containing free sulfhydryl groups in 31493 EZ Link Maleimide Activated Kit the hinge region Labeling in this area is advantageous because it directs the modification Alkaline Phosphatase Kit away from the antigen binding region Native proteins lacking a free sulfhydryl on their sur Includes EZ Link Maleimide amg i Activated Alkaline Phosphatase face can be reacted with SATA to generate blocked sulfhydryl groups The SATA molecule Activation Conjugation Buffer 20 ml reacts with primary amines via its NHS ester end to form stable amide linkages The acety BupH Tris Buffered Saline Pack 2 packs lated sulfhydryl group blocked is stable until treated with hydroxylamine to generate the d a VPage free sulfhydryls Polyacrylamide Desalting Column 1x10 ml mE l MercaptoethylamineeHCl 6 mg Pierce offers stable preactivated enzyme derivatives that are reactive toward sulfhydryl SATA 2 mg SH groups EZ Link Maleimide Activated Alkaline Phosphatase Product 31486 and s a Horseradish Peroxidase Product 31485 These products eliminate the first step of the Column Extender two step maleimide method simplifying and facilitating the conjugation protocol while sav 31485 EZ Link Maleimide Activated 5 mg ing several hours They can be used to prepare enzyme conjugates dir
93. munoconjugates and Radiopharmaceuticals 2 37 46 Wordinger R J et al 1987 Manual of Immunoperoxidase Techniques 2nd Edition Chicago American Society of Clinical Pathologists Press pp 23 24 Yolken R H 1982 Rev Infect Dis 4 1 35 68 Alkaline Phosphatase A highly sensitive enzyme for ELISA and immunohistochemical applications Highlights e Purified form ready to conjugate without prior dialysis Activity is not affected by exposure to antibacterial agents such as sodium azide or thimerosal e Specific activity gt 2 000 units mg e One unit is defined as the amount that will hydrolyze 1 0 umole of p nitrophenyl phos phate per minute at 37 C in 1 0 M diethanolamine 0 5 mM MgCl pH 7 8 Specific Activity per mg Protein Buffer 25 C 37 C 0 1 M Glycine 1 0 mM ZnCl 1 0 mM MgCl gt 500 gt 1 000 6 0 mM p Nitrophenyl phosphate pH 10 4 1 0 M Diethanolamine 0 5 mM MgCl gt 1 000 gt 2 000 15 mM p Nitrophenyl phosphate pH 9 8 References Bulman A S and Heyderman E 1981 J Clin Pathol 34 1349 1351 Cordell J L et al 1984 J Histochem Cytochem 32 219 229 Yolken R H 1982 Rev Infect Dis 4 35 68 PRODUCT DESCRIPTION PKG SIZE 31490 ImmunoPure Horseradish Peroxidase 10 mg 31491 ImmunoPure Horseradish Peroxidase 100 mg 31391 ImmunoPure Alkaline Phosphatase 20 mg Calf intestinal Supplied in Tris Buffer pH 7 Triethanolamine 1 mM MgCL 3 M NaCl pH 7 6 31392
94. n of hydrogen peroxide and because fewer preparation steps are involved it provides more consistent results Although the Stable Peroxide Substrate Buffer is provided as a 10X concentrate it is also stable at a 1X concentration PRODUCT DESCRIPTION PKG SIZE 34062 Stable Peroxide Buffer 10X 100 ml o 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 1 Step TMB Blotting Product 34018 is a single compo nent peroxidase substrate for Western blotting and immunohistochemistry Precipitating the product results in dark blue bands where the enzyme is located 1 Step TMB Blotting is well suited to applications that require a large Signal to noise ratio PRODUCT DESCRIPTION PKG SIZE 34018 1 Step TMB Blotting 200 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 PRODUCT DESCRIPTION PKG SIZE 34012 1 Step CN 250 ml 34010 4 Chloro 1 Napthol Powder 25 g powder 34011 4 Chloro 1 Napthol Tablets 50 tablets DAB has a molecular weight of 214 1 and y
95. n the optimal image Erase It amp Technology can be used for any appli cation using X ray film exposures including e Western Northern and Southern blots such as with SuperSignal Substrates and the North2South Chemiluminescent Detection Kit e n gel detection systems e Gel shift assays e Ribonuclease protection assays RPA PRODUCT DESCRIPTION PKG SIZE 21065 Erase It Background Eliminator Kit Sufficient reagent to prepare 3 liters of working solution Includes Erase It Reagent A 100 ml Erase It Reagent B 100 ml Featured Product con t Lightens Overexposed Bands After using Erase It Solution Before using Erase It Solution Figure 33 Recombinant human wild type p53 baculovirus lysate was separated on a 12 SDS polyacry lamide 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 mem brane was exposed to film for 1 minute A The film had overexposed bands and was treated with Erase It Background Eliminator for 6 minutes The resulting image provided better visualization of the different p53 protein bands B Before Erase It Eliminator 6 1 Minute lt 2 5 Minutes O 4 Minutes Relative Intensity 1 000 250 62 5 15 625 pg sample Figure 34 D
96. nd divalent cation chelators such as EDTA 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 a longer time period Horseradish peroxidase is a 40 000 dalton pro tein that catalyzes the oxidation of substrates by hydrogen peroxide resulting in a colored or flu orescent product or the release of light as a 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 conjugates 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 2 Comparison of horseradish peroxidase and alkaline phosphatase enzymes Horseradish Alkaline Peroxidase Phosphatase Size 40 kDa 140 kDa Price Relatively Relatively Inexpensive Expensive Stability Stable at lt 0 C Unstable at 0 C Storage Number of Many Few Substrates Kinetics Rapid Slower pH optimum 5 7 8 10 Tel 800 874 3723 or 815 968 0747 www piercenet com Affinity Purified Secondary Antibodies ImmunoPure Affinity Purified Antibodies are available unconjugated or conjugated with biotin alkaline phosphatase horseradish peroxidase fluorescein or rhod
97. new Electrophoresis and Staining Handbook from Pierce includes technical and ordering information for all the Pierce products you need to separate and detect your proteins The well organized handbook walks you E step by step through the electrophoresis process and beyond Contact Pierce your Perbio Science branch office or your local distributor to request your FREE copy of this helpful handbook www piercenet com Tel 815 968 0747 or 800 874 3723 e Fax 815 968 7316 Customer Assist Outside the United States visit our web site or call 815 968 0747 to locate your local geno Belgium amp Dist France Germany The Netherlands Tel 32 0 53 83 44 04 Tel 0800 50 82 15 Tel 0228 9125650 Tel 076 50 31 880 euroinfo perbio com euroinfo perbio com de info perbio com euroinfoGperbio com uk anch dU pute Pierce products are suppied for laboratory or manufacturinc 0 Pierce Biotechnology Inc 2004 Unless otherwise noted all trademarks belong to Pi
98. nformation on this method see page 44 Most instrument companies know and recommend SuperSignal West Substrates over other chemiluminescent substrates for use in their instruments Featured Product CL XPosure Film Save 60 75 on film t 0 Pierce Kodak BioMax CL XPosure Film MR 1 Film Figure 20 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 as indicated in the table below Kodak X Omat Blue XB Film Cost Comparison of 5 x 7 Sheets Product Cost per sheet U S Price Pierce CL XPosure Film Blue X ray Film 0 79 Kodak X Omat Blue XB Film Blue X ray Film Perkin Elmer 2 10 Kodak BioMax MR 1 Gray X ray Film Amersham 3 36 Source 2004 Online Catalogs For more product information or to download a product instruction booklet visit www piercenet com 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 instruments but sensitivity may not be as good as that which is obtained with film e magers sometimes require longer exposure tim
99. nnon J G 1985 Different blocking agents cause variation in the immunologic detection of proteins transferred to nitrocellulose membranes J Immunol Meth 81 161 Towbin H et al 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets procedure and some applications Proc 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 detection of peroxidase conjugated antibod ies on immunoblots J Virol Meth 24 227 236 SuperSignal Technology is protected by U S Patent 6 432 662 Slide A Lyzer MINI Dialysis Unit Technology is protected by U S Patent 6 039 781 U S patent pending on UnBlot Technology Bio Rad is a trademark of Bio Rad Laboratories Inc Chemilmager is a trademark of Alpha Innotech Kathon and Triton are trademarks of Rohm amp Haas LumiPhos is a trademark of and is sourced from Lumigen Inc Novex and NuPAGE are trademarks of Novel Experimental Technology BioMax and X Omat are trademarks of Kodak Tween is a registered trademark of ICI Americas ECL ECL Plus and Hybond are trademarks of Amersham Pharmacia Biotech For more product information or to download a product instruction booklet visit www piercenet com n The
100. nsfer Membranes Nitrocellulose Membranes PRODUCT DESCRIPTION PKG SIZE 88013 Nitrocellulose Membrane 0 2 um 15 pkg 7 9 cm x 10 5 cm 88018 Nitrocellulose Membrane 0 45 um 1 roll 33 cmx3m 88014 Nitrocellulose Membrane 0 45 um 15 pkg 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 68024 Nitrocellulose Membrane 0 2 um 15 pkg 8 cm x 8 cm T1012 Nitrocellulose Membrane 0 2 um 25 pkg 8 cm x 12 cm 68025 Nitrocellulose Membrane 0 45 um 15 pkg 8 cm x 8 cm 77011 Nitrocellulose Membrane 0 45 um 10 pkg 8 cm x 12 cm T1010 Nitrocellulose Membrane 0 45 um 25 pkg 8 cm x 12 cm Polyvinylidene Difluoride PVDF Membranes PRODUCT DESCRIPTION PKG SIZE 88585 Capture PVDF Transfer 10 sheets Membrane 0 45 um 10 cm x 10 cm 88518 Capture PVDF Transfer 1 roll Membrane 0 45 um 26 5 cm x 3 75 m Blocking Buffer Optimization SuperBlock Blocking Buffer Milk Casein BSA 1 50 1 10 1 2 1 50 1 10 1 2 1 50 1 10 1 2 E ca e gt Cyclin B1 30 Second Exposure p53 30 Second Exposure fos 30 Second Exposure of E fos 5 Minute Exposure Figure 6 Blocking buffer optimization Blocking Buffers Application Chart Product Blocking Buffer ELISA Western DotBlot Immunohisto DNA RNA blot chemistr Hybridizations 3 538 StartingBlock v v v v PBS Blocking Buffer 3 542 StartingBlock v v v v TBS Blocking Buffer 3 539 4 Sta
101. ntain azide as a preservative e Azide is an inhibitor of HRP do not use azide as a preservative Exposure time is too short e Lengthen the film exposure time Note SuperSignal Chemiluminescent Substrates will continue to glow for a minimum of six hours Substrate incubation is too short e A five minute substrate incubation is required when using SuperSignal Substrates The wrong membrane was used e Nitrocellulose is the recommended membrane when using SuperSignal West Pico Chemiluminescent Substrate for Western Blotting PVDF can be used but may require further optimization e SuperSignal West Dura Chemiluminescent Substrate and SuperSignal West Femto Chemiluminescent Substrate can be used with nitrocellulose PVDF nylon and charge modified nylon membranes Substrate has lost activity e SuperSignal West Pico Chemiluminescent Substrate and SuperSignal West Dura Chemiluminescent Substrate are stable for up to 12 months at RT SuperSignal West Femto Chemiluminescent Substrate is stable for at least six months at RT o determine if the substrate has lost 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 has lost activity e Ensure that there is no cross contamination between the two bottles Contamination between the two substrate reagents can cause a decline in activity Membrane h
102. o be performed 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 complete ly wetted i e approximately 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 B mercap toethanol 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 inter fere 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 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 as described above with fresh 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 followed by a wash in wash buffer Apply fresh SuperSignal West Working Solution as described above If no signal is detected with a 5 minute exposure the primary anti
103. o film for 30 seconds resulting in considerable background speckling A The film was then treated with Erase It8 Background Eliminator for 2 minutes to eliminate the background speckling B Tel 800 874 3723 or 815 968 0747 www piercenet com Blotting with Chemiluminescence Troubleshooting Guide High Background that is Uniformly Distributed Possible Causes Precautions Solutions Antibody concentrations are too high The primary and or secondary antibody can cause high background if the concentrations used are too high Decrease antibody concentrations Wrong blocking buffer was used e Compare different blocking buffers Insufficient blocking of e Optimize blocking buffer The best blocking buffer is system dependent nonspecific sites e Increase the concentration of protein 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 to blocking buffer Use a final concentration of 0 05 Tween 20 Skip this step if you use StartingBlock T20 Blocking Buffer in PBS Product 37539 or TBS Product 37543 or SuperBlock9 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 with 0 05 Tween 20 Cross reactivity of antibody with Use a different blocking buffer other proteins in blocking buffer
104. on Kit Includes PhosphoProbe HRP 2 mg EDC 9g Ethylenediamine 10g Tween 20 1 vial PhosphoProbe HRP binds only to free phosphates 0 II iP eo C N C C NH Phospho protein HoN C C N C PO3 FES une 0 Tel 800 874 3723 or 815 968 0747 www piercenet com Featured Product O GIcNAc Western Blot Detection Kit CH High specificity monoclonal against O GIcNAc The Pierce O GlcNAc Western Blot Detection Kit contains the most highly specific mouse s j monoclonal antibody available for the detection of the O GlcNAc posttranslational modifica 0H tion on a membrane Reaction of the monoclonal antibody in this Western blotting kit is HaC H HO confined to the B O linked serine or threonine GlcNAc modification There is no cross reac H i tivity with the 0 GIcNAc linkage the B 0 GalNAc modification or the other N linked E oligosaccharides HC 0 CH 0H Speed and sensitivity of chemiluminescent detection l Chemiluminescent detection with Pierce SuperSignal West Dura Extended Duration N N Substrate allows visualization of O GlcNAc modified proteins in less than one minute after H H H exposure of the blot to X ray film In addition to speed this kit is sensitive to the low pico mole range B 0 GIcNAc Modified Serine Performance validated on Jurkat cell lysates Threonine in Peptide Linkage This Western blot kit also includes our popular M PER Mammalian Cell Lysis Reagen
105. on of UnBlot Chemiluminescent Substrate optimized for both on membrane and in gel use Featured Product ProFound Far Western Protein Protein Interaction Kits Highlights e On membrane or in gel detection options on membrane detection offers greater sensitivity in gel detection method offers speed and prevents problems associated with incomplete or inefficient transfer e Nonradioactive alternative for far Western analysis reliable and sensitive biotin a haa PRODUCT DESCRIPTION PKG SIZE streptavidin HRP or anti GST H RP chemistry combined with chemiluminescent detection 23500 ProFound Far Western 10 mini gels offers a practical and safe alternative to radiolabeling the bait protein Biotinylated Protein Protein Interaction Kit e Useful interaction range kit targets moderate to strong associations between a prey and ee eee ey iotinvlated bait tei GST t d b tei in gel or on membrane of protein the biotinylated bait protein or agged probe protein interactions using a biotinylated bait protein as the probe e Primary antibody free detection kit uses a biotinylated or GST tagged protein as the Includes Streptavidin HRP 0 1 mg iminati j j Dilution Buffer 10X 50 ml probe eliminating the need for antibody production BupH Phosphate Buffered 17 packs e Compatible with both SDS PAGE and native gels provides option to probe for prey UE SUE proteins in a more native environment because reduced or
106. ontains some components that require storage at 4 C 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 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 M Immunoblot Stained Gel Figure 22 Western blot detection of 0 GlcNAc 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 BlueRanger Prestained Protein Molecular Weight Marker Mix Product 26681 For more product information or to download a product instruction booklet visit www piercenet com o Far Western Blottinc Studying Protein Interactions by Far Western Blotting Far Western blotting was originally developed to screen protein expression libraries with 32P 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 It is possible to study the effect of post translational modifications on protein protein interac
107. or SuperSignal West Substrates mix equal vol umes 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 0 125 ml cm of blot surface 11 Incubate the blot with SuperSignal9 Substrate Working Solution for 5 minutes or with Lumi Phos WB Substrate Working Solution for 3 minutes 12 Remove the blot from the substrate working solution and place it in 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 13 Place the wetted blot against the film and expose Standard autoradiographic film can be used A recommended first exposure time is 60 seconds Exposure time can be varied 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 th
108. orm ing 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 an example of a dot blot protocol using SuperSignal West Pico Substrate When using other substrates from Pierce refer to the instruction booklet for recommended antigen antibody concentrations Tel 800 874 3723 or 815 968 0747 www piercenet com Optimizing Antibody Concentration Note All antibody dilutions assume a starting concentration of 1 mg ml 1 Recommended Primary Antibody Dilutions Prepare dilutions of the protein sample in either TBS or PBS The proper dilution will depend on the antigen concentration present in the sample because the concentration of the antigen of interest often is not known It is necessary to test a wide range of dilu tions 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 immunoassay results may show any or all of the following detec tion of nonspecific bands blurred banding patterns and rapid signal deterioration Prepare nitrocellulose membranes The number of membrane pieces needed depends on how many different dilutions of primary and or secondary antibody will be screened Typically one or two dilutions of the primary antibody are tested with two or three differ ent di
109. polyhistidine tags independent of adjacent tags Highlights e Specific more specific for the detection of histidine tagged fusion proteins than anti His antibodies e Fast one step probe incubation eliminates the need to run a lengthy two step primary secondary antibody sequential reaction protocol e Sensitive when used in combination with SuperSignal West Chemiluminescent Substrates kit allows the detection of even low expression histidine tagged clones e More versatile than anti polyHis antibody based systems the HisProbe Kit detects poly histidine fusion proteins that are undetectable using some monoclonal anti polyHis antibodies e Sufficient reagents for fifty 7 5 x 10 cm blots 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 PNAS 99 19 24 Sylvester S R and Roy A 2002 Biol Reprod 67 895 899 Phosphoprotein Detection Reagent and Kit Novel chemistry enables specific detection of phosphorylated protein PhosphoProbe HRP is an iron Fe activated derivative of horseradish peroxidase HRP PhosphoProbe HRP exhibits two distinct binding specificities one of which is phosphate R PO specific The other binding specificity is related to a carboxyl containing binding motif that is common to most proteins and some peptides This carboxyl motif binding Specificity can be used in a total prot
110. rSignal West Dura Extended Duration Substrate Product s 34076 and 34075 e Lumi Phos WB Substrate Product 34150 Colorimetric Substrates e 1 Step Chloronaphthol Product 34012 e TMB Blotting Product 34018 e NBT BCIP Product 34042 e Metal Enhanced DAB Product 34065 Surfact Amps Brand Detergents containing Tween 20 Product 28320 and Tween 80 Product 28328 e Triton X 100 Product 28314 and Triton X 114 Product 28332 Nonidet P 40 Product 28324 e Brij 35 Product 28316 and Brij 58 Product 28336 For convenience and economy Pierce also offers complete Western blotting Kits that include chemiluminescent substrates enzyme conjugated antibodies blocking buffers and standard buffers Film Expose the membrane to X ray film e CL XPosure Film P D R rimary and Secondary Detection Reagents 5 x 7 sheets Product s 34090 and 34092 Incubate the membrane with antibody 8 x 10 sheets Product s 34091 and 34093 For a complete list visit the antibody selection guide on our web site www piercenet com accessible under the Products tab For direct detection methods we offer n e Monoclonal Antibodies e Fluorescent Probes and Labeling Kits e Erase It Background Eliminator Kit Product 21065 SDS Stripping Buffer e Enzyme Labeling Kits Reprobe the blot if necessary For indirect detection methods we offer Restore Western Blot Stripping Buffer
111. rane with 4 Incubate membrane with 5 Rinse membrane with Total time 15 minutes transfer with ultrapure water Reagent 1 for 2 minutes ultrapure water Reagent 2 for 10 minutes ultrapure water on a shaker repeat 5 times on a shaker repeat 5 times Figure 31 Qentix Western Blot Signal Enhancer Protocol performed after transfer and before blocking Tel 800 874 3723 or 815 968 0747 www piercenet com Featured Product Erase It Background Eliminator Another method by which the S N ratio can be improved is to erase the background leav ing just the signal with little to no interference Erase It9 Background Eliminator does just that without altering the integrity of the data The Erase It Solution works on overexposed film lightening the entire film evenly The end result is to effectively reduce the initial expo sure time This is done directly in the lab while viewing the film No darkroom is required The process can be halted when the signal is clearly visible and the background is at a mini mum thereby increasing the S N ratio without altering the data s integrity Figure 32 Erase It amp 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
112. rcenet com Fast Red TR AS MX Substrate is a sensitive Substrate that results in a bright red precipitate on transfer membranes Alkaline phosphatase hydrolyzes the naphthol phosphate ester sub Strate The naphthol then couples to the colorless chromogen which is a diazonium salt resulting in a precipitating azo dye This sub Strate is ideal for double staining in both immunoblotting and immunohistochemical Studies The bright red precipitate contrasts well with horseradish peroxidase insoluble sub Strates including CN DAB and TMB The red precipitate fades upon drying but will reappear when rehydrated with water When double Staining immunoblots the Fast Red TR AS MX solution must be added before a horseradish peroxidase substrate solution to prevent exces sive background color development PRODUCT DESCRIPTION PKG SIZE 34034 Fast Red TR AS MX Kit Includes Fast Red TR Salt 250 mg Napthol AS MX 35 ml Phosphate Concentrate 250 ml Substrate Buffer Chemiluminescent Substrates When energy in the form of light is released from a substance because of a chemical reac tion 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 15 0 0 0 uoo 425 nm NH O NHs O NH O Figure 15 Lum
113. reasons 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 conju gates such as horseradish peroxidase or alkaline phosphatase The indirect method offers many advantages over the direct method Substrate Detectable Product r Enzyme Substrate Detectable xx Product r 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 anti body and reacts with the substrate Advantages of Direct Detection Figure 1A e t is a quick methodology because only one antibody is used e Cross reactivity of secondary antibody is eliminated e Double staining is easily achieved using different labels on primary antibodies from the same host Disadvantages of Direct Detection Figure 1A e Immunoreactivity of the primary antibody may be reduced as a result of labeling e Labeling of every primary antibody is time consuming and expensive e There is no flexibility in choice of primary antibody label from one exper
114. ripping the antibody under gentle conditions Because each anti body 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 was designed to achieve maximum removal of antibodies from a mem brane while preserving the integrity of the antigen It is unique among stripping buffers because it is odor free and can often strip a membrane in as little as 15 minutes Stripping and reprobing a Western blot instead of running an entirely new blot may be preferable because it e Conserves sample When the protein mixture is rare or valuable reprobing conserves 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 Makes it easy to optimize The increased light emission intensity of SuperSignal West Pico Substrate along with 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 blots membrane buffers and
115. rot 1 5 mg 1 5 ml 1 5 mg 1 5 ml 1 ml Rabbit IgG F ab Goat 31234 31923 31573 31461 31343 2 mg 2 ml 2 mg 2 ml 1 ml Rabbit IgG Fc Goat 31216 31463 31341 am 2 ml 1 ml Rabbit IgG H L Mouse 31213 31824 31584 31674 31464 min x GtHnMsSh Sr Prot 1 5 mg 1 ml 1 mg 1 mg 1 ml Anti RABBIT Rabbit IgG H L Goat 31214 31579 F ab Fragment 1 mg 1 mg of Host Antibody Rabbit IgG H L Goat 31215 min x Hn Sr Prot 1 mg Rabbit IgG Fc Goat 31217 31581 1m 1m Anti RAT Rat IgG H L Goat 31220 31930 31629 316980 31470 31350 2 mg 2 ml 2 mg 2 mg 2 ml 1 ml Rat IgG F ab Goat 31474 2 ml Rat IgG Fc Goat 31226 31833 31621 31475 31353 2 mg 2 ml 2 mg 2 ml 1 ml Rat IgM p Goat 31228 31832 31631 31476 31354 2 mg 2 ml 2 mg 2 ml 1 ml Rat IgG H L Rabbit 31218 31834 2 mg 1 5 mg Rat IgG H L Rabbit 31219 31936 min x Ms Sr Prot 0 5 mg 0 5 mg Anti RAT Rat IgG H L Rabbit 31227 F ab Fragment 1 mg of Host Antibody Rat IgG IgM Goat 31625 H L 1m Rat IgG H L Mouse 31225 31633 31682 min x Ms Sr Prot 1m 0 5m 0 5m Anti SHEEP sheep IgG H L Rabbit 31240 31840 31627 31480 31360 2m 1 5m 1 5m 1 5 ml 1 ml Sheep IgG Fc Rabbit 31241 31841 31441 31356 2m 1 5 ml 1 5 ml 1 ml Sheep IgG F ab Rabbit 31244 31844 31481 31344 2 mg 1 5 ml 1 5 ml 1 ml Anti SHEEP Sheep IgG H L Rabbit 31229 F ab Fragment 1 mg of Host Antibod Anti SWINE swine IgG H L Goat 31231 2 mg See Table 3 on page 12 for the Key to Abbreviations Tel 800 874 3723
116. rtingBlock T20 v v v v PBS Blocking Buffer 3 543 StartingBlock T20 v v v v TBS Blocking Buffer 3 515 SuperBlocko v v v v v Blocking Buffer in PBS 3 535 SuperBlock v v v v v Blocking Buffer in TBS 3 517 SuperBlocke v v v Blocking Buffer Blotting in PBS 3 53 SuperBlocko v v v Blocking Buffer Blotting in TBS 3 516 SuperBlock v v v v v T 20 PBS Blocking Buffer 3 536 SuperBlock9 v v v v v T 20 TBS Blocking Buffer 37527 SEA BLOCK v v v Blocking Buffer 3 520 Blocker BSA v v v v v in TBS 3 525 Blocker BSA v v v v v in PBS 3 532 Blocker v v v v v Casein in TBS 3 528 Blocker v v v v v Casein in PBS 3 530 Blocker v v v v v BLOTTO in TBS These blocking buffers are recommended for use when performing Western blots with SuperSignal Chemiluminescent Substrates 1 50 1 10 1 2 Blocking Buffer Optimization The most appropriate blocking buffer for Western blotting use is often system depend ent Determining the proper blocking buffer can help to increase the system s signal to noise ratio Occasionally when switching from one Substrate to another the blocking buffer that you are using will lead to diminished signal or increased background Empirically testing vari ous blocking buffers with your system can help achieve the best possible results Avoid using milk as a blocking reagent for blots that rely on the avidin biotin system because milk contains variable amounts of
117. s Rinse four times with ultrapure H 0 Wash on a shaker with ultrapure H O for 5 minutes I O N WW C Stain Erasing Protocol 1 Wash with MemCode Stain Eraser on a shaker for 2 minutes 2 Rinse four times with ultrapure H 0 3 Wash with ultrapure H O on a shaker for 5 minutes Rinse three times with MemCode Destain Solution A PVDF Membrane Staining Protocol 1 Wash membrane with ultrapure H 0 2 Add MemCode Sensitizer Shake for 2 minutes 3 Add MemCode Stain Shake for 1 minute Protein bands appear turquoise in color B Destaining Protocol 1 Rinse three times with MemCode Destain Solution 2 Wash with Memcode Destain mixed 1 1 with MeOH on a shaker for 5 minutes 3 Rinse five times with ultrapure H O C Stain Erasing Protocol 1 Wash with MemCode Stain Eraser mixed 1 1 with MeOH on a shaker for 10 20 minutes 2 Rinse five times with ultrapure H O Tel 800 874 3723 or 815 968 0747 www piercenet com Blocking Nonspecific Binding Sites 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 vari ety of blocking buffers ranging from milk or normal serum to highly purified proteins have been used to block unreacted sites on a membrane The blocking buffer should improve the sensitivity of the assay by reducing background interference Individu
118. s 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 solutions are covered 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 the new Qentix Western Blot Signal Enhancer Qentix 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 The Qentix Western Blot Signal Enhancer membrane treatment is a simple 15 minute pro cedure 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 with the Western Blot Signal Enhancer compared to the typical detection protocol without treatment Untreated blot Blot treated with Qentix Western Blot Enhancer 1 2 3 4 1 2 3 4 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 what substrate is used
119. sult in unsatisfactory results For more product information or to download a product instruction booklet visit www piercenet com Featured Product SuperSignal West Pico Chemiluminescent Substrate Twice as much signal for about 40 less than the price of the 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 ECL System Supersignal West Pico Chemiluminescent Substrate costs much less than the ECL System SuperSignal West Pico ECL Western Blotting Detection Substrate Product 34080 Substrate Product RPN2106 2004 U S List Price 0 05 0 07 per cm of membrane More stable Supersignal West Pico Substrate is room temperature RT stable for months with no dis cernable 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 expo sure conditions In most cases there is no need to rerun samples and repeat the blotting procedure Highlights e Economy costs less per ml than other chemi luminescent substrates e Longer light emission strong light emission over a working day allows you to make several exposures e High intensity signal is twice as intense as o
120. t an HRP labeled anti IgM antibody conjugate blocking buffer and wash buffer components all validated to perform as specified Highlights Kit includes MAb CTD 110 6 the most specific monoclonal antibody for the detection of B PRODUCT DESCRIPTION PKG SIZE 24565 0 GIcNAc Western Blot Kit Detection Kit Sufficient material to develop up to 10 mini blots O linked M acetylglucosamine O GIcNAc Includes M PER Mammalian 25 ml TON T Protein Extraction Reagent Dilution e Detect only the B O GlcNAc modification on the proteins within the sample Buffer 10X Blocking Buffer 2x50m ifingti Nii BupH Phosphate Buffered Saline 17 packs e Detection of the target modification confined to only B O linked serine or threonine Surfac Amps 20 m e No cross reactivity with the 0 GIcNAc linkage 10 Tween 20 solution Anti O GlcNAc Monoclonal Antibody 100 ul TE i MAb CTD 110 6 in ascites Kit includes M PER9 Mammalian Protein Extraction Reagent Goat ant Mouse IgM 75g e Convenient efficient eukaryotic cell lysis HRP Conjugate SuperSignal West Dura Extended 100 ml Kit includes super sensitive patented SuperSignal West Dura Chemiluminescent Substrate Duration Substrate e Provides maximum sensitivity from the antigen primary secondary HRP complex formed at the site of a B O GIcNAc modification e Sensitivity to low picomole level Note This Western blot kit is shipped in a single box as a two part kit Part A c
121. te Femto Substrate Dura Substrate WB Substrate Recommended Nitrocellulose Nitrocellulose Nitrocellulose Nitrocellulose Membrane or PVDF or PVDF or PVDF 5 Remove the membrane blot and block the nonspecific sites with a blocking buffer for 20 60 minutes at RT with Shaking For best results block for 1 hour at RT Optimization of blocking buffer may be required to achieve best results Please see the Optimization of Blotting Buffers section page 7 6 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 Optimization of Antibody Concentration section page 22 SuperSignal West SuperSignal West SuperSignal West Lumi Phos Pico Substrate Femto Substrate Dura Substrate WB Substrate Recommended 1 1 000 1 5 000 1 5 000 1 100 000 1 1 000 1 50 000 1 200 1 2 000 Primary Antibody or 0 2 1 0 ug ml or 0 01 0 2 ug ml or 0 02 1 0 ug ml or 0 5 5 0 ug ml Dilutions from 1 mg ml stock Wash the membrane with wash buffer At least four to six changes of the wash buffer are recommended Use as large a volume of wash buffer as possible 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
122. tern Analysis Gel Electrophoresis Native or denaturing usually Native usually or denaturing Transfer System Optimal membrane and transfer system determined empirically Optimal membrane and transfer system determined empirically Blocking Buffer Optimal blocking system determined empirically Optimal blocking system determined empirically Detection Unlabeled primary antibody Unlabeled bait protein several possible Enzyme labeled secondary antibody Enzyme labeled bait specific antibody Strategies substrate reagent Substrate reagent Enzyme labeled primary antibody Arrows designate oubstrate reagent sequence of steps Biotinylated antibody in the detection Enzyme labeled streptavidin strategy Substrate reagent Radiolabeled bait protein Exposure to film Biotinylated bait protein Enzyme labeled streptavidin substrate reagent Fusion tagged bait protein Tag specific antibody Enzyme labeled secondary antibody oubstrate reagent Labeled antibodies generally are enzyme labeled either horseradish peroxidase or alkaline phosphatase By contrast bait proteins generally are not enzyme labeled because a large enzyme label is likely to sterically hinder unknown binding sites between bait and prey proteins Other labeling and detection schemes are possible Tel 800 874 3723 or 815 968 0747 www piercenet com Far Western Blotting Critical Steps in Far Western Analysis Gel Electrophoresis Separation of proteins by SDS PA
123. tes Pour off the wash buffer and repeat Brief rinses of the mem branes before incubation in the wash buffer may increase the wash step efficiency Prepare dilutions of the secondary antibody HRP conjugate 1 20 000 1 100 000 in blocker Tween 20 Add the secondary antibody dilutions to the membranes and incu bate for 1 hour with shaking SuperSignal SuperSignal SuperSignal Lumi Phos West Pico West Femto West Dura WB Substrate Substrate Substrate Substrate Recommended Secondary 1 20 000 1 100 000 1 100 000 1 500 000 1 50 000 1 250 000 1 5 000 1 25 000 Antibody Dilutions or 10 50 ng ml or 2 0 10 ng ml or 4 0 20 ng ml or 40 200 ng ml from 1 mg ml stock 8 J Wash the membrane again as described in Step 6 Prepare the substrate working solution by mixing equal volumes of the Luminol Enhancer Solution and the Stable Peroxide Solution Prepare a sufficient vol ume to ensure that the blot is completely wetted with substrate and the blot does not dry out during incubation Recommended volume 0 125 ml cm of blot surface instruction booklet visit www piercenet com For more product information or to download a product 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 An
124. ther compatibly priced luminol based systems e Picogram sensitivity highly sensitive for the rapid development of a wide range of protein levels e Excellent stability 24 hour plus working solu tion stability kit is stable for at least one year at ambient temperature e Saves antibody primary and secondary anti bodies are diluted further so they can be used for more blots Enhanced Light Emission Kinetics SuperSignal Substrate vs ECL System 500 000 400 000 300 000 200 000 Net Relative Intensity 100 000 0 SuperSignal ECL System West Pico Substrate Figure 17 Net relative intensity six hours after incubation Supersignal West Pico Substrate 1 minute 50 25 125 6 3 3 1 16 08 04 02 0 1 05 03 013 006 003 ng ass ECL System 1 minute 90 25 12563 31 1 6 08 0 4 0 2 0 1 05 03 013 006 003 ng Supersignal West Pico Substrate ECL System 9 minutes 9 minutes 3062 519156 3 3 1 GeO 8e 0 4 02 0 1 205 03 2 038 0858003 ng 90 25 12563 3 1 1 6 0 8 0 4 0 2 0 1 05 03 013 006 003 ng c Die PLN Figure 16 50 ng of Recombinant Mouse IL 2 was serially diluted down to 0 003 ng and electrophoresis was performed The gels were trans ferred 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 aga
125. tion and then not deviate from the method Binding and Wash Conditions Protein protein interactions vary depending on the nature of the interacting proteins The strength of the interactions may depend on the pH salt concentrations and the presence of certain co factors during incubation with the bait protein Some protein protein interactions may also require the presence of additional proteins Whatever the necessary conditions they will need to be maintained throughout the procedure to maintain the interaction until it can be detected This may influence the formulation of washing buffer used between prob ing steps instruction booklet visit www piercenet com For more product information or to download a product Controls When identifying protein protein interactions by the far Western technique it is important to always include appropriate controls to distin guish true protein protein interaction bands from nonspecific artifactual ones For example experiments involving detection with recombi nant GST fusion proteins should be replicated with GST alone A bait protein with a mutation in the predicted interaction domain can be processed as a control to determine specificity of the protein protein interaction A non relevant protein can be processed along Side the prey protein sample to act as a negative control Ideally the control protein would be of similar size and charge to the protein under investigation and wou
126. units of a multi subunit complex By an ordered fragment lad der far Western analysis they were able to identify the interaction domains of F coli RNA polymerase B subunit The protein was expressed as a polyhistidine tagged fusion then partially cleaved and purified using a Ni chelate affinity column The polyhistidine tagged fragments were separated by SDS PAGE and transferred to a nitrocellulose membrane The fragment localized interaction domain was identified using a P labeled protein probe Table 10 Comparison of Western blotting and far Western blotting methods Importance of Native Prey Protein Structure in Far Western Analysis Far Western blotting procedures must be per formed with care and attention to preserving as much as possible the native conformation and interaction conditions for the proteins under study Denatured proteins may not be able to interact resulting in a failure to identify an inter action Alternatively proteins presented in non native conformations may interact in novel artificial ways resulting in false positive interac tions The prey protein in particular is subjected to preparative processing steps for far Western blotting that can have significant effects on detec tion of protein protein interactions This is not to imply that identification of valid interactions is not possible but only to stress the importance of appropriate validation and use of controls Step Western Blotting Far Wes
127. use of block ing buffer or inappropriate enzyme labeled probe or antibody concentration Overexposed bands are a common occurrence when the enzyme labeled probe or antibody concentration used is too high or if the film was exposed for too long Speckling and shading occur when enzyme conjugates form complexes and precipitate on the blot The Erase It9 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 32 33 and 35 The Erase It9 Solution can be used with newly exposed films or exposed films that have been stored for years In addition the Erase It amp Kit can be used with any brand of film For applications requiring densitometric measurement the Erase It Background Eliminator reduces signal evenly over the film so that relative densitometry values are consistent Figure 34 The procedure is simple Immerse your exposed film in Erase It Working Solution watch for desired image and stop the reaction by rinsing the film in water The Erase It Solution works quickly with ideal signal level typically attained in just a few minutes A Overexposed Film S Old option New option Start over and re optimize antibody Use Erase It Background Eliminator concentration and blocking buffer B Two days later C Four minutes later Figure 32 A431 cell lysate was electrophoresed on a 4 12 NuPage Gel Novex an
128. 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 Figure 13 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 OH CH3 CH E SU OCG OO OC CH CH I TMB 4 CN DAB M W 240 4 M W 178 6 M W 214 1 Figure 12 Chromogenic substrates for Western blotting with HRP OCH OCH H NBT BCIP M W 817 6 M W 433 6 Naphthol AS MX phosphate Fast Red TR Salt CH N coed OX td Cl Fast Red TR AS MX Substrate Figure 13 Chromogenic substrates for Western blotting with AP 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 prior to use As a result these substrates typically have a useful shelf life of only a few hours Many precipitating HRP substrates from Pierce are supplied 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 solutio
129. ves immunodetection of proteins directly in the gel This technique Pierce UnBlot In Gel Detection circumvents the transfer and blocking steps entirely allowing immunoblotting techniques to be applied to proteins that cannot be trans ferred efficiently from a gel to a membrane Because there is no transfer of proteins from gel to membrane no protein is lost in the process and no artifacts are introduced into the data This makes UnBlot Detection an ideal control experiment to confirm results obtained by Western blotting and to study proteins that cannot be transferred to a membrane Another feature of the UnBlot System is that it does not require any blocking step If there is no blocking then there is no 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 Protein left in a gel after transfer to a nitrocellulose membrane 12 3 45 678 9 10111213 14 Figure 23 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 Rad9 Mini Gel Transfer Unit Following the transfer the protein left in the gel was detected using the UnBlot 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
130. 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 man ufacturer s recommended dilution Horseradish peroxidase 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 above In blot 1 the blot was totally black due to both the primary and secondary anti body 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 blots 3 nor 4 had background sig nal Primary Antibody 1 500 Secondary Antibody 1 5 000 Primary Antibody 1 5 000 Secondary Antibody 1 50 000 Figure 11 Example of signal intensity on a Western blot using SuperSignal West Dura Substrate and antibodies at various concentrations In Figure 11 blots were optimized with SuperSignal West Dura Chemiluminescent substrate The blot with a primary antibody concentration of 1 500 and a secondary anti body concentration of 1 5 000 shows what happens when the antibody levels are too high The background is not excessively high but the bands are too intense and blur together resulting in poor reso
131. within 6 hours The Sulfo NHS ester group reacts with primary amines on the enzyme surface to form a stable amide bond After this first step of con jugation the enzyme will have maleimide groups on its surface that react optimally toward sulfhydryl groups between pH 6 5 and 7 5 to form stable thioether bonds Maleimide mediated conjugation strategies are summarized in Figure 8 Tel 800 874 3723 or 815 968 0747 www piercenet com Labeling Your Own Antibodies Three methods for free sulfhydryl generation Maleimide activation of enzyme Native protein contains disulfide bonds that can be reduced to generate free sulfhydryls Enzyme is SMCC labeled through primary amines to generate a maleimide activated enzyme for conjugation to free sulfhydryls S S J ES SN E HN EDA 2020 H 4 0 Ny Oo D Native protein has a free 3 Native protein is reacted with SATA Blocked sulfhydryl SMCC sulfhydryl on its surface groups are introduced on primary amines Hydroxylamine H treatment generates free sulfhydryls SH Sy HO N D oe E CCH the B l 4 Q C CH SH NHS 4 Figure 8 Three strategies for maleimide mediated conjugation of enzymes Two reagents MercaptoethylamineeHCI Product 20408 and SATA Product 26102 are available to produce free sulfhydryls on macromolecules for conjugation to the maleimide PRODUCTS DESCRIPTION ___________FKG ME activated enzymes For labeling antibody
132. y standard or enhanced autoradiographic film can be used A recom mended first exposure is 30 60 seconds Exposure time can be varied to obtain opti mum 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 Substrate 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 Chromogenic Substrates As with the other components in a Western blotting system there are many substrate choices available The appropriate substrate choice depends on the enzyme label AP or HRP desired sensitivity and desired form of signal or method of detection Chromo genic substrates have been employed most widely 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 visualizing Substrates such as TMB 3 3 5 5 tetramethyl benzidine 4 CN 4 chloro 1 naphthol and DAB 3 3 diaminobenzidine tetrahydrochloride are available for use with HRP Figure 12 For
133. y 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 demonstrates high background problems e Thimerosal free formulation PRODUCT DESCRIPTION PKG SIZE 37532 Blocker Casein in TBS 1 liter 1 w v Casein Hammersten Grade in TBS Contains Kathon Antimicrobial Reagent as preservative pH 7 4 37528 Blocker Casein in PBS 1 liter 1 w v Casein Hammersten Grade in PBS Contains Kathon Antimicrobial Reagent as preservative pH 7 4 Blocker BLOTTO Ready to use blocking buffers made of nonfat dry milk Highlights e Preformulated for ease of use e Anti foaming agent added e Available in TBS Buffer e Merthiolate free formulation PRODUCT DESCRIPTION PKG SIZE 37530 Blocker BLOTTO in TBS 1 liter 5 w v nonfat powdered milk in TBS 0 01 Anti foam A contains Kathon Antimicrobial Reagent as preservative pH 7 4 Blocker BSA For all blocking applications Highlights e 10 solutions of high quality Bovine Serum Albumin e Concentrated formulation saves storage Space e No waiting for powder to dissolve with this ready to dilute liquid concentrate PRODUCT DESCRIPTION PKG SIZE 3 925 Blocker BSA in PBS 10X 200 ml 3 920 Blocker BSA in TBS 10X 125 ml Surfact Amps 20 Purified Detergent Solution Specially purified form of Tween 20 Highlights e Guaranteed lt 1 milliequivalent

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