For more product information, or to download a productinstruction book - PDF document

For more product information, or to download a productinstruction booklet, visit www.piercenet.coml: 800-874-3723 or 815-968-0747www.piercenet.com Grasp the Proteomeª WesternHandbook and Troubleshooting GuideSuperSignalFamily of Products For more product information, or to download a productinstruction booklet, visit www.piercenet.comIntroduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1estern Blotting Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3ransfer Protein to a Membrane. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4MemCodeª Protein Stains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-5Blocking Nonspecific Binding Sites. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6ransfer Buffers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6ransfer Membranes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Blocking Buffer Optimization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Blocking Buffers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-9ashing the Membrane. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10ash Buffers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Primary and Secondary Antibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Conjugate Stabilizer Solutions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12Affinity-Purified Secondary Antibodies. . . . . . . . . . . . . . . . . . . . . . . . 13-15Labeling Your Own Antibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Enzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17EZ-Linkª Activated Enzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18-19EZ-Labelª Fluorescent Labeling Kits. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Optimizing Antibody Concentration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21-22Chromogenic Substrates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23-24Chemiluminescent Substrates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25-31Chemiluminescent Substrates. . . . . . . . . . . . . . . . . . . . 26-30LumiPhosª Chemiluminescent Substrate. . . . . . . . . . . . . . . . . . . . . . . . 31Quick Reference Substrate Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Data Imaging. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32CL-XPosureª Film. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32estern Blot Detection Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34Far-Western Blotting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35-36In-Gel Western Detection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37-39Optimizing the Signal-to-Noise Ratio. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40Restoreª Western Blot Stripping Buffer. . . . . . . . . . . . . . . . . . . . . . . . . 42Qentixª Western Blot Signal Enhancer. . . . . . . . . . . . . . . . . . . . . . . . . . 43Background Eliminator. . . . . . . . . . . . . . . . . . . . . . . . . . . . 44-45roubleshooting Guide Ð Blotting with Chemiluminescence. . . . . . . . . . . . . 46-49estern Blotting Protocol Using Chemiluminescent Substrates. . . . . . . . . . 50-51Recommended Reading. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 l: 800-874-3723 or 815-968-0747www.piercenet.com 12341234A.MemCodeB.Ponceau S StainStain 12341234A. ControlB. MemCode 1234567891012345678910A. MemCodeStain B. Ponceau S StainReversible Protein Stain with Ponceau S stain on PVDF mem-Unstained Protein M.W. Markers (Product # 26671) were serially diluted andapplied to two 4-20% Tris-glycine-SDS polyacrylamide gels Lanes 1-9was stained with 0.1% Ponceau S in 5% acetic acid for 5 minutes and destained according tothe published protocol. Prestained M.W. Marker Mix (Product # 26681). A.Nitrocellulose Membrane Staining ProtocolA.PVDF Membrane Staining Protocol1.Wash membrane with ultrapure HO.1.Wash membrane with ultrapure H2.Add MemCodeª Stain. Shake 30 seconds.2.Add MemCodeª Sensitizer. Shake for 2 minutes.Protein bands appear turquoise in color. 3.Add MemCodeª Stain. Shake for 1 minute.Protein bands appear turquoise in color.B.Destaining ProtocolB.Destaining Protocol1.Rinse three times with MemCodeª Destain Solution.1.Rinse three times with MemCodeª Destain Solution.2.Add MemCodeª Destain. Shake 5 minutes.2.Wash with Memcodeª Destain mixed 1:1 with 3.Rinse four times with ultrapure H3.Rinse five times with ultrapure H4.Wash on a shaker with ultrapure HC.Stain Erasing ProtocolC.Stain Erasing Protocol1.Wash with MemCodeª Stain Eraser on a shaker 1.Wash with MemCodeª Stain Eraser mixed 1:1 withfor 2 minutes.MeOH on a shaker for 10-20 minutes.2.Rinse four times with ultrapure HO.2.Rinse five times with ultrapure H3.Wash with ultrapure H Increasing amounts of GST Lysate protein were applied ontotwo 4-20% Tris-glycine SDS polyacrylamide gels. Both gelstreated with MemCodeaccording to the protocol. Blot Ponceau S stain for 5 minutes and destained. The blot stainedbands. GST Lysate loading volumes (Lane 1-3). Lane 1: 5 µl,Lane 2: 10 µl, Lane 3: 15 µl, Lane 4: BlueRanger purifiedGST applied to two 10% Tris-glycine SDS polyacrylamide gels. Bothtrol membrane (Panel anti-rabbitIgG-HRP conjugate and detected using Pierce(Product # 34075).Lane 1:125 pg, Lane 2: 250 pg, Lane 3: 500 pg and Lane 4: 1 ng. PRODUCT #DESCRIPTIONSIZEProteinStain Kit for reverse the stain from 10 (8 cm x 8 cm) Includes: MemCodeReversible Stain250 mlA broad-spectrum stain for proteins Destain*1,000 mlStain Eraser*500 ml reverse the stain from 10 (8 cm x 8 cm) Includes: MemCodeSensitizer250 mlReversible Stain250 mlA broad-spectrum stain for proteins Destain*1,000 mlStain Eraser*500 ml *Reagent-grade methanol (required, but not supplied) For more product information, or to download a productinstruction booklet, visit www.piercenet.comansfer BuffersBupHª Tris-Glycine and Tris Buffered SalineGreat for Western blots! BupHª Tris-Glycine Buffer PacksEach pack yields 500 ml of 25 mM Tris and 192BupHª Tris Buffered Saline PacksEach pack yields 500 ml of 25 mM Tris, 0.15 MNaCl, pH 7.2 when dissolved in 500 ml deion- In a Western blot, it is important to block the unreacted sites on the membrane to reducethe amount of nonspecific binding of proteins during subsequent steps in the assay. A vari-sensitivity of the assay by reducing background interference. Individual blocking buffers arenot compatible with every system. For this reason, a variety of blockers in both Tris bufferedbuffer in TBS should be selected because PBS interferes with alkaline phosphatase. TheFor true optimization of the blocking step for a particular immunoassay, empirical testing isimmunoassay, it is important to test several different blockers for the highest signal-to-noiseratio in the assay. No single blocking agent is ideal for every occasion because each anti-Pierce offers a complete line of blocking buffers for Western blotting including BLOTTO, Featured Products PRODUCT #DESCRIPTIONPKG. SIZE BupHª Tris-Glycine Buffer Packs BupHª Tris Buffered Saline Packs BupHª Tris Buffered Saline Packs PRODUCT #DESCRIPTIONPKG. SIZENitrocellulose Membrane, 0.2 µm Nitrocellulose Membrane, 0.45 µm 33 cm x 3 mNitrocellulose Membrane, 0.45 µmMinimum 87 sheets when cut to 7.9 cm x 10.5 cm; minimum 52 sheets when cut to Nitrocellulose Membrane, 0.2 µm Nitrocellulose Membrane, 0.2 µm Nitrocellulose Membrane, 0.45 µm Nitrocellulose Membrane, 0.45 µm Nitrocellulose Membrane, 0.45 µm 8 cm x 12 cm PRODUCT #DESCRIPTIONPKG. SIZECaptureª PVDF TransferMembrane, 0.45 µm Captureª PVDF TransferMembrane, 0.45 µm 26.5 cm x 3.75 m For more product information, or to download a productinstruction booklet, visit www.piercenet.com Like other immunoassay procedures, Western blotting consists of a series of incubationswith different immunochemical reagents separated by wash steps. Washing steps are nec-essary to remove unbound reagents and reduce background, thereby increasing thefrom the blot. As with other steps in performing a Western blot, a variety of buffers may beused. 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% Tweendetergent in wash buffers helps to minimize background in the assay. For best results, usehigh-purity detergents, such as Surfact-AmpsDetergents for Western blotting.BupHª Dry Buffers1.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 thereÕs no waste.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.1.BupHª Buffers are protected from contamination and are fresh every time. 2.Carry out applications with confidence in buffer quality.3.ÒTest-assuredÓ with the Pierce commitment to quality management standards.1.Making routine buffers is no longer time-consuming.2.No component measurement, pH adjustment, quality validation, preparation tracking or3.Move forward with your work by eliminating re-tests due to buffer problems. Great wash buffer for Western blots!0.15 M NaCl, pH 7.0 when dissolved in 500 ml PRODUCT #DESCRIPTIONPKG. SIZEBupHª Phosphate Buffered BupHª Tris Buffered SalineGreat wash buffer for Western blots! Each pack yields 500 ml of 25 mM Tris, 0.15 MNaCl, pH 7.2 when dissolved in 500 ml deion- PRODUCT #DESCRIPTIONPKG. SIZEBupHª Tris-Glycine BupHª Tris Buffered BupHª Tris Buffered Specially purified form of Tweenimprove performance PRODUCT #DESCRIPTIONPKG. SIZE 28320Surfact-Amps6 x 10 ml For more product information, or to download a productinstruction booklet, visit www.piercenet.com able 3: Key to abbreviations for individual species Bv = BovineGu = Guinea PigHs = HorseRt = Rat Ch = ChickenHa = HamsterMs = MouseSh = Sheep Gt = GoatHn = HumanRb = RabbitSw = Swineer. Table 4 lists the typical working dilutions for the conjugated antibodies when doingable 4: Typical dilution ranges recommended for Pierce ImmunoPure Conjugated Antibodies ConjugateELISAImmunoblottingImmunohistochemistryAlkaline 1:5,000-1:50,0001:2,500-1:25,0001:500-1:5,000 Peroxidase1:5,000-1:200,000 1:25,000-1:500,0001:500-1:5,000 ELISA Products)West Products) FluoresceinÑÑ1:50-1:200 RhodamineÑÑ1:50-1:200 Affinity-Purified Secondary AntibodiesUsing Antibodies: A Laboratory Manualin bioresearch likeAntibodies: A LaboratoryThe authors, however,Using Antibodies: A Laboratory Manualand handling it correctly, to the proper methodsworking with antibodies and antigens, Usingtion of theory, methods and results. The bookThis helpful guide, along with high-quality prod-ucts from Pierce, will help you purify, immobilize,label and store antibodies and perform commonbetween the Fc portions of antibodies and Fc receptor-bearing cells must be eliminated.ies, resulting in an increase in sensitivity and specificity, with a decrease in background. Thepurification process involves an elution procedure, yielding antibodies with high avidity.Prot.Ó The key to abbreviations for the individual species is shown in Table 3.Pierce provides a variety of reagents to help preserve enzyme conjugate activity. Typically,conjugates are aliquoted in 50-100 µl increments using purified ethylene glycol (Product #29810) as a preservative for -20¡C storage. Conjugates can maintain activity for up to twoto one year as a stock solution. Guardianª Peroxidase Stabilizer/Diluents (Product #Õs PRODUCT #DESCRIPTION Guardianª Peroxidase Conjugate Stabilizer/Diluent (SD) Guardianª Peroxidase Conjugate Stabilizer/Diluent (SD) SuperFreezeª Peroxidase Conjugate Stabilizer (50% aqueous solution) PRODUCT #DESCRIPTIONPKG. SIZEA Laboratory ManualLaboratory Press, 1999. 495 pages; * Sorry, books are nonreturnable. For more product information, or to download a productinstruction booklet, visit www.piercenet.com Affinity-Purified Secondary Antibodies *See Table 3 on page 12 for the Key to Abbreviations. Pkg. Size SpecificityDescriptionHostUnconj.Biotin-LCFluoresceinRhodaminePeroxidaseAlk. Phos.Anti-HUMANHuman IgG (H+L)Mouse3113731784 continued(min x BvHsMs Sr Prot)*1.5 mg1 mlHuman IgG (H+L)Rabbit3114331786 2 mg1.5 mlHuman IgG (H+L)Rabbit31147 (min x Ms Sr Prot)*1.5 mgHuman IgG (Fc)Rabbit3114231789315353142331318 2 mg1.5 ml1.5 mg1.5 ml1 mlHuman IgM (Fc5µ)Rabbit31149 Anti-HUMANHuman IgG (Fc)Goat31163Fragment 1 mg Human IgG (H+L)Goat31165 Human IgA + IgG Goat31539 + IgM (H+L)1 mgHuman IgGMouse31155 (min x MsBvHs Sr Prot)*1.5 mgAnti-MOUSEMouse IgA (Goat31169 (min x Hn Sr Prot)*1 mgMouse IgA + IgGGoat31171 + IgM (H+L)2 mgMouse IgG (H+L)Goat311603180031569316603143031320 2 mg2 ml2 mg2 mg2 ml1 mlMouse IgG (H+L)Goat311643180231541316613143231322 (min x BvHnHs Sr Prot)*1.5 mg1.5 mg1.5 mg1.5 mg1.5 ml1 ml1 ml()2]Goat3116631803315433143631324 2 mg2 ml2 mg2 ml1 mlMouse IgG (Fc)Goat311683180531547316633143731325 2 mg2 ml2 mg2 mg2 ml1 mlMouse IgG (Fc)Goat311703143931327 (min x BvHnHs Sr Prot)*1.5 mg1.5 ml1 mlMouse IgM (µ)Goat311723180431992316623144031326 2 mg0.5 mg2 mg2 mg2 ml1 mlMouse IgM (µ)Goat311763158531664 (min x BvHnHs Sr Prot)*1.5 mg1.5 mg1.5 mgMouse IgG + IgMGoat3118231807315863144431328 (H+L)2 mg2 ml1.5 mg2 ml1 mlMouse IgG + IgMGoat311843144631330 (H+L) (min x BvHnHs Sr Prot)*1.5 mg1.5 ml1 mlMouse IgG (H+L)Horse3118131806 1.5 mg1.5 mgMouse IgG (H+L)Rabbit311883181031561316653145031329 2 mg1.5 ml1.5 mg1.5 mg1.5 ml1 mlMouse IgG (H+L)Rabbit31190318123145231334 (min x Hn Sr Prot)*1.5 mg1 ml1 ml1 ml()2]Rabbit311923181131559316663145131331 2 mg1.5 ml1.5 mg1.5 mg/1.5 ml1 mlMouse IgG (Fc)Rabbit3119431813315553145531332 2 mg1.5 ml1.5 mg1.5 ml1 mlMouse IgM (µ)Rabbit3119631814315573145631333 2 mg1.5 ml1.5 mg1.5 ml1 mlMouse IgG + IgMRabbit3119831815315583145731335 (H+L)2 mg1.5 ml1.5 mg1.5 ml1 mlMouse IgG (H+L)Goat311853156531438 (min x BvHnHs Sr Prot)*1 mg1 mg0.5 ml For more product information, or to download a productinstruction booklet, visit www.piercenet.comChemical cross-linking reagents have become an invaluable tool in the scientific community.can be prepared from a ligand of interest, then reacted with a primary amine on the surfaceof the enzyme. While this method is necessary in some applications, such as those in whichthe ligand does not contain a primary amine, it is not useful as a general-purpose method. CarbohydrateCarbohydrate S-S S-S S-S S-S S-S PapainPepsin S-S S-S 2 FC S Heavy Chains VHAmines onlysine residuesSulfhydryls created whenantibody is reduced enzymes to create reagents for Western blot-targeting. Pierce offers tools for a varietyPrimary aminesdant and distributed over the entire antibody.Sulfhydryl groupsbody. Labeling Your Own Antibodies For more product information, or to download a productinstruction booklet, visit www.piercenet.comwo reagents, Mercaptoethylamine¥HCl (Product # 20408) and SATA (Product # 26102), areavailable to produce free sulfhydryls on macromolecules for conjugation to the maleimide-activated enzymes. For labeling antibody molecules, mild reduction with Mercaptoethyl-amine¥HCl (MEA) results in two half-antibody fragments containing free sulfhydryl groups inaway from the antigen-binding region. Native proteins lacking a free sulfhydryl on their sur-face can be reacted with SATA to generate blocked sulfhydryl groups. The SATA moleculereacts with primary amines via its NHS ester end to form stable amide linkages. The acety-lated sulfhydryl group (blocked) is stable until treated with hydroxylamine to generate thefree sulfhydryls.Pierce offers stable, preactivated enzyme derivatives that are reactive toward sulfhydryl peptides or other ligands that contain a free ÐSH group. Two reagents, SATA and mercap-toethylamine¥HCl, are also included in the kit formats to produce free sulfhydryls onChoi, J.Y., Seo, Y.R., oo, J.H., SH Protein Protein Protein Three methods for free sulfhydryl generationMaleimide activation of enzymeNative protein contains disulfide bonds that can be reduced to generate free sulfhydryls.Enzyme is SMCC labeled through primary S S SH Protein SATA Protein Protein SO C H Protein Protein NOC HN OOOO O O NNHS OH O N N +H2N O O N O N O N O N Figure 8. Three strategies for maleimide-mediated conjugation of enzymes. Labeling Your Own Antibodies PRODUCT #DESCRIPTIONPKG. SIZE Includes: EZ-LinkMaleimide 2 mgActivation/Conjugation Buffer20 mlBupHª Tris Buffered Saline Pack2 packsBupHª Phosphate Buffered1 packPolyacrylamide Desalting Column1 x 10 mlMercaptoethylamine¥HCl6 mgSATA2 mgHydroxylamine5 mgDMF1 ml Column Extender31485EZ-LinkMaleimide Activated5 mg Includes: EZ-LinkMaleimide 5 mgActivated Horseradish Peroxidase20 ml2-Mercaptoethylamine¥HCl6 mgSATA2 mgDimethylformamide1 mlHydroxylamine¥HCl5 mg Polyacrylamide Desalting Column1 x 10 ml For more product information, or to download a productinstruction booklet, visit www.piercenet.com Featured ProductEZ-Labelª Fluorescent Labeling KitsEZ-Labelª Kits contain everything you need to successfully label your antibody or protein:Slide-A-LyzerExcitationEmissionEZ-Labelª KitWavelength (nm)Wavelength (nm)Fluorescein Protein Labeling Kit491518Rhodamine Protein Labeling Kit544576 Fluorescein Isothiocyanate494520These kits contain sufficient reagents to perform five fluorescent labeling reactions, which use up to 10mg/ml of protein for each reaction (50 µl-1 ml volume of protein). Step 1. Preparation of ProteinFor salt-free lyophilized protein:Dissolve in borate buffer.Reconstitute fluorescent Add dye to the protein solution. Step 2. Labeling ReactionStep 3. Removal of Excess Fluorescent Dye See how easy the fluorescent labeling procedure is when you use EZ-Labelª Kits. PRODUCT #DESCRIPTIONPKG. SIZEIncludes: No-Weighª 6 x 1 mgPre-Measured Fluorescein microtubesDimethylformamide (DMF)1 mlBupHª Borate Buffer Packs5 packsBupHª Phosphate Buffered 5 packsD-Saltª Dextran Desalting 5 columnsSlide-A-LyzerMINI Dialysis 5 units Reaction Tubes5 tubesIncludes: No-Weighª 6 x 0.5 mgPre-Measured Rhodamine microtubesDimethylformamide (DMF)1 mlBupHª Borate Buffer Packs5 packsBupHª Phosphate Buffered 5 packsD-Saltª Dextran Desalting 5 columnsSlide-A-LyzerMINI Dialysis 5 units Reaction Tubes5 tubesIncludes: No-Weighª 6 x 1 mgDimethylformamide (DMF)1 mlBupHª Borate Buffer Packs5 packsBupHª Phosphate Buffered 5 packsD-Saltª Dextran Desalting 5 columnsSlide-A-LyzerMINI Dialysis 5 units Reaction Tubes5 tubes l: 800-874-3723 or 815-968-0747www.piercenet.com Figure 10. Example of signal intensity on a Western blot when using SuperSignalRecombinant Human Wild-Type p53 Baculovirus lysate at variousufacturerÕs recommended dilution. Horseradish peroxidase-labeled goat anti-mouse was added at differentalso 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 oftoo much primary and secondary antibody. In blots 3 and 4, the signal-to-noise was much better because boththe primary and secondary antibody concentrations were reduced. Neither blots 3 nor 4 had background sig-Figure 11. Example of signal intensity on a Western blot using SuperSignalSubstrate. The blot with a primary antibody concentration of 1:500 and a secondary anti-The background is not excessively high, but the bands are too intense and blur together,mal blot was achieved by using a 1:5,000 primary antibody dilution and a 1:50,000secondary antibody dilution.Because every new Western blot is unique,there is no "perfect" antibody concentration forevery blot. Therefore, every new Western blottemÕs key components (see page 7).activity of both the primary and secondary anti-body will vary. The optimal antigen and antibodyconcentrations can be determined by perform-ing complete Western blots with varyingconcentrations of antigen and antibody.Alternatively, a faster and easier method is toperform a dot blot procedure. The following isother substrates from Pierce, refer to the Mouse anti-Human p531/5001/5001/1,0001/1,000(1 µg/ml)(1 µg/ml)(0.5 µg/ml)(0.5 µg/ml)Goat anti-Mouse HRP1/1,0001/5,0001/10,0001/20,000(1 µg/ml)(0.2 µg/ml)(0.1 µg/ml)(0.05 µg/ml)Exposure Time30 seconds30 seconds1 minute1 minute Primary Antibody 1:500Secondary Antibody 1:5,000Primary Antibody 1:5,000Secondary Antibody 1:50,0001.2.3.4. For more product information, or to download a productinstruction booklet, visit www.piercenet.com 1.Prepare dilutions of the protein sample in either TBS or PBS. The proper dilution willthe antigen of interest often is not known. It is necessary to test a wide range of dilu-2.Prepare nitrocellulose membranes. The number of membrane pieces needed depends onhow many different dilutions of primary and/or secondary antibody will be screened.ypically, one or two dilutions of the primary antibody are tested with two or three differ-ent dilutions of the secondary antibody. For example: 1/1,000 primary with 1/50,000secondary, 1/1,000 primary with 1/100,000 secondary, 1/5,000 primary with 1/50,000secondary, and 1/5,000 primary with 1/100,000 secondary.3.Place dry membranes. Apply the smallest possible volume to the membranes (2-5 µl works well)the antigen dilutions to dry on the membranes for 10-30 minutes or until no visible4.Block the nonspecific sites on the nitrocellulose membranes by incubating them inblocking buffer that contains 0.05% Tween-20 (blocker/Tween-20) for 1 hour at RT5.Prepare the primary antibody dilutions (1/1,000-1/5,000) in blocker/Tweenapply to the membranes. Incubate for 1 hour at RT with shaking.est PicoWest FemtoWest DuraWB SubstrateSubstrateSubstrateSubstrateRecommended Primary1/1,000-1/5,000 1/5,000-1/100,000 1/1,000-1/50,0001/200-1/2,000or 0.2-1.0 µg/mlor 0.01-0.2 µg/mlor 0.02-1.0 µg/mlor 0.5-5.0 µg/ml(from 1 mg/ml stock)6.Wash the membrane 4-6 times in TBS or PBS, using as large a volume of wash bufferas possible. Add 0.05% Tweenbranes before incubation in the wash buffer may increase the wash step efficiency.7.Prepare dilutions of the secondary antibody/HRP conjugate (1/20,000-1/100,000) inblocker/Tween-20. Add the secondary antibody dilutions to the membranes and incu-est PicoWest FemtoWest DuraWB SubstrateSubstrateSubstrateSubstrateRecommended Secondary1/20,000-1/100,000 1/100,000-1/500,000 1/50,000-1/250,0001/5,000-1/25,000or 10-50 ng/mlor 2.0-10 ng/mlor 4.0-20 ng/mlor 40-200 ng/ml(from 1 mg/ml stock)8.Wash the membrane again as described in Step 6.9.Prepare the substrate working solution by mixing equal volumes of thedry out during incubation. 10.Incubate the membrane in the SuperSignalest Pico Substrate Working Solution for 511.Remove the membrane from the substrate12.Place the blot against the film Ð protein sidemum results. Alternatively, use a CCDcamera or other imaging device; however,13.On an optimized blot, the SuperSignalnecessary as the blot ages. If optimal results For more product information, or to download a productinstruction booklet, visit www.piercenet.com Chromogenic Substrates Br CI P alkalinephosphatase Br CI OH HPO- H H CI CI O CI H Odichloro-indigo white-2H NBTNBT-formazan pounds known as tetrazolium salts. Upon reduction, the compound yields NBT-formazan, ahighly colored, water-insoluble product. The substrate is widely used for immunochemical PRODUCT #DESCRIPTIONPKG. SIZE Nitro-Blue Tetrazolium Chloride PRODUCT #DESCRIPTIONPKG. SIZE BCIP. Together, they yield an intense, black-purple precipitate that provides much greateraccurate control of its relative sensitivity. Figure 14. Reaction of alkaline phosphatase with BCIP and NBT. PRODUCT #DESCRIPTIONPKG. SIZE 1-Stepª NBT/BCIP 1-Stepª NBT/BCIP Plus Suppressorprecipitate fades upon drying but will reappearwhen rehydrated with water. When double- PRODUCT #DESCRIPTIONPKG. SIZEFast Red TR/AS-MXIncludes: Fast Red TR Salt250 mgNapthol AS-MX35 mlPhosphate Concentrate 250 ml Substrate Buffer For more product information, or to download a productinstruction booklet, visit www.piercenet.comand Western Lightningª Products est PicoECLSubstrate Cost ComparisonSubstrateSubstrateSubstrate Membrane (8 x 10 or 8 x 12)$ 5.66 $ 8.30 $12.40 TBS Wash Buffer$ 1.24 $ 1.24 $ 1.30 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 otal Blotting Cost$19.49 $53.73 $34.64 *EndogenÕs Anti-CD54 (Product # MA5407, 500 µg) was used at the substrate manufacturerÕs recommended starting dilution.Costs are based on January 2004 U.S. list prices for an 8 x 10 cm mini gel following manufacturerÕs instructions. est Pico SuperSignalest DuraSuperSignalChemiluminescent SubstrateExtended Duration SubstrateMaximum Sensitivity SubstratePrimary Benefitwice the signal for about half ¥ Extended signal duration is¥ The most sensitive chemiluminescentthe price of competing productsideal for use with imaging equipmentsubstrate for HRP detection availableLow-picogram (10Low-femtogram (106-8 Hours¥ 24 Hours¥ 8 HoursSuggested Antibody Dilutions**Primary: 1/1,000-1/5,000¥ Primary: 1/1,000-1/50,000¥ Primary: 1/5,000-1/100,000Secondary: 1/20,000-1/100,000¥ Secondary: 1/50,000-1/250,000¥ Secondary: 1/100,000-1/500,000Room Temperature (RT) ¥ 24 Hours¥ 24 Hours¥ 8 Hours¥ 1 year at RT¥1 year at RT¥ 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. SuperSignalSubstrates are much more sensitive than other substrates, so it is critical that you follow these guidelines.Failure to do so could result in unsatisfactory results. Chemiluminescent Substrates For more product information, or to download a productinstruction booklet, visit www.piercenet.com Featured Product PRODUCT #DESCRIPTIONPKG. SIZEChemiluminescent SubstrateIncludes: Luminol/Enhancer250 ml Stable Peroxide Buffer250 mlChemiluminescent SubstrateIncludes: Luminol/Enhancer2 x 25 ml Stable Peroxide Buffer2 x 25 mlChemiluminescent SubstrateIncludes: Luminol/Enhancer25 ml Stable Peroxide Buffer25 mla complete Western blotting kit! IgG or NeutrAvidinª Biotin-Binding Protein PRODUCT #DESCRIPTIONPKG. SIZEest Pico Complete est Pico Mouse IgGDetection Kitest Pico Complete est Pico Rabbit IgGDetection Kitest Pico Complete Biotinylated Protein Detection Kit34085SuperSignalWest PicoKit Biotinylated Protein DetectionKit l: 800-874-3723 or 815-968-0747www.piercenet.com Featured Product imaging instruments. It is increasingly common to see these instruments in todayÕs researchlabs. Pierce developed SuperSignalneeds of researchers using this efficient new technology. Cooled CCD cameras, which offerthe advantages of instant image manipulation, higher sensitivity, greater resolution and adarkroom. However, this technology has one drawback Ð it requires a substrate that pro-by the cameras. Pierce developed SuperSignalcombining 24-hour light emission with ultraintensity, SuperSignalallows researchers to take full advantage of all the features offered by imaging instruments. erÕs recommended dilutions and membrane. ECL Plusª Substrate strongly recommends theuse of PVDF. The proteins were transferred to PVDF for the ECL Plusª Substrate and to nitro-System. The primary antibody for both substrates was used at aA 10 ng/ml dilution was used for the secondary antibody SuperSignal 24-hour light emission Ð 10 times longer thanother chemiluminescent substrates; performComes with HRP-labeled secondary antibodiesyear with ambient shipping conditions ECLPlusª Substrate 30 minutes (ChemiImagerª 4000) 15 minutes (ChemiImagerª 4000) Figure 17. 50 ng of recombinant mouse IL-2 was serially diluted to 0.003 ng andest Duraand then incubated with a 1 µg/ml dilution of the primary antibody, rat anti-mouse IL-2. After washing, the membranes were incubated with the secondary antibody,pared according to the manufacturerÕs instructions. Each membrane was exposed toX-ray film for 5 minutes. The SuperSignalexposed to the ChemiImagerª 4000 for 30 minutes and the ECL Plusª Blot wasexposed for 15 minutes. (The exposure of the ECL Plusª Blot was not extended to 30minutes due to the high background that had already accumulated at 15 minutes.) PRODUCT #DESCRIPTIONPKG. SIZEIncludes: Luminol/Enhancer50 mlStable Peroxide Buffer50 mlHRP-Conjugated Goat Anti-Rabbit1 ml HRP-Conjugated Goat Anti-Mouse1 mlIncludes: Luminol/Enhancer100 mlStable Peroxide Buffer100 mlHRP-Conjugated Goat Anti-Rabbit1 ml HRP-Conjugated Goat Anti-Mouse1 mlest Dura ExtendedDuration Substrate Trial KitIncludes: Luminol/Enhancer10 ml Stable Peroxide Buffer10 ml l: 800-874-3723 or 815-968-0747www.piercenet.comFeatured Product Lumi-Phosª WB Chemiluminescent SubstrateSDS-polyacrylamide gel.were washed and then incubated in Lumi-Phosª WB Substrate for five minutes prior to film exposure.Lumi-Phosª WB Chemiluminescent Substrate overcomes all of the limitations posed byconventional chemiluminescent substrates for AP.background noise than other popular chemiluminescent substrates for AP, providing a bettersignal:noise ratio and a clearer image. Because signal generation is immediate, thereÕs nowith a low, low price. Dilution range of AntibodyApproximate SubstrateProduct #Measurement / Color(From 1 mg/ml stock)Sensitivity*Enzymeest Pico Substrate34080425 nm chemiluminescent1¡1:1K-1:5K1 pgHRP 2¡1:20K-100Kest Dura Substrate34075425 nm chemiluminescent1¡1:1K-1:50K250 fgHRP 2¡1:50K-250Kest Femto Substrate34095425 nm chemiluminescent1¡1:5K-1:50K60 fgHRP 2¡1:100K-500K1-Stepª TMB - Blotting Substrate34018Dark blue PPT1¡1:5001 ngHRP 2¡1:2K-20K1-Stepª 4-CN Substrate34012Blue-purple PPT1¡1:5001 ngHRP 2¡1:2K-20KCN/DAB Substrate34000Black PPT1¡1:5001 ngHRP 2¡1:2K-20KDAB Substrate34001Brown PPT1¡1:5001 ngHRP 2¡1:2K-20KMetal Enhanced DAB Substrate34065Brown-black PPT1¡1:50020 pgHRP 2¡1:2K-20KLumi-Phosª Substrate34150440 nm chemiluminescent1¡1:5K15 pgAP 2¡1:25K1-Stepª NBT/BCIP Substrate34042Black-purple PPT1¡1:50030 pgAP 2¡1:2.5K1-Stepª NBT/BCIP + Suppressor Substrate34070Black-Purple PPT1¡1:500 30 pgAP 2¡1:2.5KNBT Substrate34035Blue-purple PPT1¡1:250100 pgAP 2¡1:2.5KBCIP Substrate 34040Blue-purple PPT1¡1:250100 pgAP 2¡1:2.5KFast Red Substrate34034Red PPT1¡1:250320 pgAP 2¡1:2.5K* Actual sensitivity is unique to each antibody-antigen pair. The approximate sensitivities listed are conservative amounts tha1¡= Primary, 2¡= Secondary, PPT = precipitate, HRP = horseradish peroxidase, AP = alkaline phosphataseLiu, R.Y., ikhonov, I., there is no need to purchase additionalAttomole-range detection and crystal-clear PRODUCT #DESCRIPTIONPKG. SIZELumi-Phosª WB Chemiluminescent Substrate For more product information, or to download a productinstruction booklet, visit www.piercenet.comCritical Steps in Far-Western AnalysisSeparation of proteins by SDS-PAGE (i.e., denaturing conditions with or without a reducingprotein and destroy or sterically hinder the interaction site on the protein. For Far-Westernstructure comprising an intact interaction site. Generally, a significant percentage of the pro-more easily detected by far-Western 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/renaturationstep 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-After transferring proteins to the membrane, Western blotting procedures require that unre-all blotting experiments. Any given protein blocker may cross-react or otherwise disrupt themust be made empirically. Often, bovine serum albumin (BSA) is used as a starting point forBinding and Wash ConditionsProtein:protein interactions vary, depending on the nature of the interacting proteins. Themay also require the presence of additional proteins. Whatever the necessary conditions, Far-Western Blottingthe far-Western technique, it is important tocontrol. Ideally, the control protein would be ofIn approaches that use a secondary system forcorresponding antibody, it is critical to probebinding of the labeled secondary antibody. To For more product information, or to download a productinstruction booklet, visit www.piercenet.com In-Gel Western Detectionreagents (antibodies, etc.) than are proteins within polyacrylamide gels. A recent advance inthe field of Western blotting involves immunodetection of proteins directly in the gel. Thistechnique, Pierce UnBlotIn-Gel Detection, circumvents the transfer and blocking stepsentirely, allowing immunoblotting techniques to be applied to proteins that cannot be trans-by Western blotting and to study proteins that cannot be transferred to a membrane.System is that it does not require any blocking step.If thereis no blocking, then there is no chance of cross-reactivity with the blocking buffer.Thissaves time because no blocking buffer optimization is necessary and background is oftenlower than with traditional Western blotting.Figure 23.Pure GFP/6xHis-tagged protein and by SDS-PAGE (Novex10-20% Tris-Glycine gels).Mini Gel Transfer Unit. Following the transfer, the protein left in the gel was detected using thegoat anti-mouse antibody. E. colibacterial GFP/6xHis-tagged lysate diluted 1:100, 1:250, 1:1,000,1:2,000 and 1:4,000, respectively. 0.5, 0.1 and 0.05 ng, respectively. 1234567891011121314 l: 800-874-3723 or 815-968-0747www.piercenet.comIn-Gel Chemiluminescent DetectionWhen performing transfers, low molecular weight (MW) proteins transfer more efficientlyAfter immunodetection, the gel can be used for total protein staining; thereÕs no need toFigure 25. Versatility and specificity of the UnBlotE. colibacterial GFP/6xHis-tagged lysate.Pure GFP/6xHis-tagged and yeast GFP extract were separated by SDS-PAGE. Both gels were pre-treatedwith 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 UnBlotSubstrate. Lanes 1, 2 and 3 correspond to 10, 5 and 1 ng pure GFP/6xHis-tagged, respectively. Lanes 4E. colibacterial GFP lysate diluted 1:100 and 1:1,000, respectively. Lanes 6 and 7 cor-respond to yeast GFP lysate 1:10 and 1:100, respectively. In-Gel Chemiluminescent Detection Kit has been tested successfully with Novexand Bio-Rad Criterionª brand gels.In-Gel Chemiluminescent Detection Kit does not perform well with Bio-Rad Ready Gels, Preciseª ProteinGels or Gradipore iGels. Studies showed 25 times lower sensitivity and require individual optimization.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.echnology 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 UnBlotchnology. In-gel DetectionMembrane Detectionseparated 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. Antigenswere detected using a 1:1,000 dilution of polyclonal Anti-Living Color Peptide Antibody, Rabbit (Clontech)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. colibacterial GFP lysate diluted 1:100 and 1:1,000, respectively. Lanes 6 and 7 correspond to yeast GFP Lysatediluted 1:10 and 1:100, respectively. Desai, S., Dworecki, B. and Cichon, E. (2001). Desai, S., Dworecki, B. and Cichon, E. (2002).Immunodetection of proteins within polyacrylamide gels.Scientific Publishing Co., pp. 413-416.Roberts, K.P., Featured Product PRODUCT #DESCRIPTIONPKG. SIZEIn-Gel ChemiluminescentSufficient reagents to perform 10 mini-gel detections.Includes: UnBlotSubstrate110 mlStabilized Goat anti-Rabbit-HRP10 µlDilution Buffer50 mlBupHª Pack PBS Buffer17 packs-205 x 10 ml Hands-Offª Incubation Colander1 unitPre-cut Cellophane10 sheets CL-XPosureª Film (5" x 7")25 sheetsIn-Gel ChemiluminescentProduct # 33500 except it contains anti-Rabbit-HRP10 µlIn-Gel ChemiluminescentIncludes: Streptavidin-HRP0.1 mgDilution Buffer50 mlPhosphate Buffered Saline17 packs10% TweenSubstrate110 ml Cellophane Exposure Sheets10 packIn-Gel ChemiluminescentDetection Kit for GST-Tagged ProteinsIncludes: Anti-Glutathione 0.25 mgS-transferase (GST)-HRPDilution Buffer50 mlPhosphate Buffered Saline17 packs10% TweenSubstrate110 ml Cellophane Exposure Sheets10 pack Substrate Hands-Offª Incubation Colander For more product information, or to download a productinstruction booklet, visit www.piercenet.comestern blotting.In Western blotting, the signal is the density of the specific protein bandoften more important than increasing the sensitivity of the system.The sensitivity of theThe General Troubleshooting Guide in the next section contains many tips on optimizing theantibody concentration.This process is made much easier by stripping and reprobing thecausing a significant amount of antigen to be released from the membrane. Various proto-every antibody while preserving the antigen. Restoreª Western Blot Stripping Bufferbrane while preserving the integrity of the antigen. It is unique among stripping buffersbecause 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 beConserves sampleWhen the protein mixture is rare or valuable, reprobing conserves the sample andIt is time-consuming to run an SDS-polyacrylamide gel and then transfer the proteinsoccur. By stripping the membrane, the blot can be reused. Optimizing the Signal-to-Noise Ratio agent, incubated with secondary antibody, thenthe primary antibody was effectively removedby the stripping procedure, no secondary anti-body should bind to the membrane and nostripping process. However, it is sometimesnecessary to alter the composition of the strip-ping buffer or change methods entirely. l: 800-874-3723 or 815-968-0747www.piercenet.comIncreasing the Sensitivity of a Western Blot 1. Rinse membrane after2. Incubate membrane with3. Rinse membrane with 1 UltrapureH2O UltrapureH2O 4. Incubate membrane with 2 Tot Figure 29. Enhanced chemiluminescent detection of identical serial dilutions of IL-6 before and aftertreatment with Qentixª Western Blot Signal Enhancer. [ken«-tiks] Western Blot Signal Enhancer ItÕs like intensifying screens in a bottle.There are many ways to increase the sensitivity of a Western blot. Some methods are assuch as optimizing antibody titer or checking for proper protein transfer.Those solutions areOne of the more certain and easiest ways to increase the sensitivity of any Western blot is touse the new Qentixª Western Blot Signal Enhancer.Qentixª Western Blot Signal Enhancer does for enzyme-/substrate-based blotting whatThe Qentixª 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 anincrease in the intensity of target protein bands on the Western blot or detection of targetin a 10-fold increase in band intensity after treatment with the Western Blot Signal EnhancerBlot treated with Qentixª Western Blot EnhancerFigure 31. Qentixª Western Blot Signal Enhancer Protocol Ð performed after transfer and before blocking. Figure 30. Enhanced chromogenic detection of identical serial dilutions of IL-6 before and after treat-ment with Qentixª Western Blot Signal Enhancer.Blot treated with Qentixª Western Blot Enhancer 12341234reatment with Western Blot Signal Enhancer canboost the band intensity from three- to 10-fold,orks with the most commonly used Western blot-B, BRCA1 and EGFNo thawing, formulating or diluting necessary* Signal enhancement of proteins on PVDF membrane hasbeen shown to be variable from no significant enhancement PRODUCT #DESCRIPTIONPKG. SIZEQentixª Western BlotSignal Enhancer* 10 cm x 10 cm blots.Includes: Enhancer Reagent 1250 ml Enhancer Reagent 2250 ml l: 800-874-3723 or 815-968-0747www.piercenet.com A.B,Figure 33.Recombinant human wild-type p53 baculovirus lysate was separated on a 12% SDS-polyacry-anti-Mouse-HRP (Product # 31434) and SuperSignalbrane was exposed to film for 1 minute (A). The film had overexposed bands and was treated with Erase-It A.B.Figure 35.Recombinant Human TNFphoresed on a 4-20% SDS-polyacrylamide gel and# 34075). The blot was exposed to film for 30 seconds,The film was then treated with Erase-It Featured Product (conÕt) After using Erase-ItBefore usingErase-It ,00062.525015.625 1 Minute Figure 34.Densitometry data on dot blot comparing before and after use of the Erase-ItEliminator.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(Product # 37515) and incubated with a 1/50,000 dilution of SA-HRP (Product # 21126). The blot was thenfilm (Product # 34092) for 5 minutes. The resulting film had high background that was cut into four stripsstrips at 1, 2.5 and 4 minutes, leaving a control strip for comparison. After scanning on a densitometer, theSolution maintaining similar slopes on a dose response curve. After using Erase-ItBefore using Erase-It l: 800-874-3723 or 815-968-0747www.piercenet.com Possible CausesPrecautions/SolutionsThe primary and/or secondary antibody can cause high background if the concentrations used Filter the conjugate through a 0.2 µm filter. ¥Use a fresh, high-quality conjugate. Optimize blocking buffer. The best blocking buffer is system-dependent. Increase concentration of protein in the blocking buffer.Optimize blocking time and/or temperature. Block for at least 1 hour at RT or overnight at 4¡C.Add Tween-20 to blocking buffer. A concentration of 0.05% TweenT20 Blocking Buffer in PBS (Product # 37516) or TBS (Product # 37536). These buffers already contain Tween Make up antibody dilutions in blocking buffer with 0.05% TweenUse a different blocking buffer. est for cross-reactivity. Block a clean piece of membrane, incubate with antibodies and then detect with Membrane was not wettedproperlyet membrane according to the manufacturerÕs instructions.Do not handle membrane with bare hands. Always wear clean gloves or use forceps.Make sure the membrane is covered with a sufficient amount of liquid at all times to prevent it from drying. ¥Handle membranes carefully Ð damage to the membrane can cause nonspecific binding.Contamination in buffers¥Use new buffers. 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. Blotting with Chemiluminescence Ð Troubleshooting Guide High Background that is Blotchy or Speckled l: 800-874-3723 or 815-968-0747www.piercenet.com Possible CausesPrecautions/Solutions ash blots after transfer. ¥Do not use SDS during immunoassay procedure. Possible CausesPrecautions/Solutions oo much protein is loaded Possible CausesPrecautions/Solutions and the appearance of white bands are indications that there is too much HRP in the system. Possible CausesPrecautions/SolutionsMake sure there are no air bubbles between the gel and membrane during transfer.et membrane according to the manufacturerÕs instructions.Do not handle the membrane with bare hands. Always wear clean gloves or use forceps. another during incubations. Nonspecific Bands Diffuse Bands Problem: Partly developed area or blank areas Blotting with Chemiluminescence Ð Troubleshooting Guide l: 800-874-3723 or 815-968-0747www.piercenet.com 8.Incubate the blot with enzyme-conjugated secondary antibody or avidin for 1 hour with shaking at RTFor recom-mended antibody- or avidin-conjugate dilutions, see the table below. The necessary dilution will vary depending onthe enzyme conjugate used, the primary antibody used in Step 6 and the amount of antigen that was transferred.estSuperSignalPico SubstrateFemto SubstrateDura SubstrateWB Substrate1/20,000-1/100,000 1/100,000-1/500,000 1/50,000-1/250,0001/5,000-1/25,000Secondary Antibodyor 10-50 ng/mlor 2.0-10 ng/ml or 4.0-20 ng/mlor 40-200 ng/ml9.Repeat Step 4 to wash away any unbound enzyme-conjugated secondary antibody. It is crucial to thoroughly washIf the working solution has not been prepared, prepare it now. For SuperSignalthe blot is completely wetted with substrate and the blot does not dry out. Lumi-Phosª WB Substrate is provided ina ready-to-use format, but it should be brought to room temperature. Substrate Working Solution for 5 minutes or with Lumi-Phosª WB SubstrateRemove the blot from the substrate working solution and place it in a plastic membrane protector. (A plastic sheetprotector works very well, although plastic wrap may also be used.) Remove all air bubbles between the blot andthe surface of the membrane protector.Place the wetted blot against the film and expose. Standard autoradiographic film can be used. A recommended firstexposure time is 60 seconds. Exposure time can be varied to obtain optimum results. The use of enhanced or pre-flashed autoradiographic film is unnecessary.If a cooled CCD Camera (e.g., Alpha Innotech CorporationÕs ChemiImagerª Camera) is used, longer exposuretimes may be necessary.Develop the film using appropriate developing solution and fixative for the type of film used.n an optimized blot, the light generated should last a minimum of six hours. The blot can be re-exposed to film, asneeded, to obtain the optimal results. Longer exposure times may be necessary as the blot ages. Pierce includes technical and ordering information forall the Pierce products you need to separate and detectbeyond. Contact Pierce, your Perbio Science branch www.piercenet.com