Thermo Scientific Pierce Western Blotting Troubleshooting Poster
Contents
1. e N r b a i j id i y p bs 7 Tips to better Western blots and better data thermoscientific com Western Possible Causes Precautions Solutions Proteins did not transfer properly e After transfer stain the gel with a total protein stain to determine transfer efficiency to the membrane Note Total protein stains may not be able to detect low quantities of antigen e Use Pierce Reversible Membrane Stain to check membrane for transfer efficiency e Ensure sufficient contact between the gel and membrane during transfer e Make sure the transfer sandwich is assembled correctly e Wet membrane according to manufacturer s instructions e Make sure transfer unit does not overheat during electroblotting procedure e Use positive control and or molecular weight markers e Optimize transfer time and current e Use Thermo Scientific Pierce Lane Marker Sample Buffer The tracking dye transfers to the membrane e Make sure sample preparation conditions have not destroyed antigenicity of the sample Note Some proteins cannot be run under reducing conditions Insufficient binding to membrane e Add 20 methanol to the transfer buffer to help binding Low MW antigen may pass through the membrane Use a membrane with a smaller pore size Insufficient amount of e Increase antibody concentrations Antibody may have poor affinity for the target protein antibodies e Antibody may have lost activity Perform a dot bl2. PI39001 and LDS Sample Buffer PI84788 e Molecular Weight Protein Ladders Thermo Scientific SuperSignal Molecular Weight Ladders s PI84785 PI84786 provide reliable and proportional band intensities in stained gels and immunoblots developed with chemiluminescent fluorescent chromogenic or other detection systems See our full line of prestained and unstained protein ladders at thermoscientific com protein ladder e Blotter the Thermo Scientific Pierce G2 Fast Blotter PI62288 can transfer proteins in as few as 5 to 10 minutes when used with our Thermo Scientific Pierce 1 Step Transfer Buffer e Transfer Buffers and Accessories we offer a variety of transfer buffers and accessories to help your blot look its best Our transfer buffers include Methanol free Transfer Buffer PI35040 Tris Glycine Transfer Buffer PI28380 and our 1 Step Transfer Buffer PI84731 The Thermo Scientific Pierce Reversible Protein Stain Kit s PI24585 PI25480 offers a non destructive reversible reliable and sensitive method to stain and detect proteins on nitrocellulose and PVDF membranes The Thermo Scientific Supersignal Western Blot Enhancer PI46640 helps increase signal to noise ratio by reducing any background noise caused by dirty primary antibodies for better sensitivity Transfer Membranes and Filter Paper our transfer membranes are available in
3. b do more see more learn more Thermo SCIENTIFIC ol Q si ol Q fecal Gel Electrophoresis The first step to a successful Western blot is to separate the proteins in your sample using gel electrophoresis We carry a full line of pre cast gels that make sample loading easier run more quickly and give you excellent resolution of your proteins Their one year shelf life and compatibility with a range of gel tanks make them the clear choice for your lab Protein Transfer After electrophoresis transfer protein from the gel to a membrane using electrophoretic transfer This step is critical to ensure that the protein adheres to the membrane The unique Thermo Scientific Pierce G2 Fast Blotter allows for the fast and efficient transfer of proteins ranging from 10 300kDa in as few as 10 minutes Consider using our signal enhancers to increase the sensitivity of your Western blot after this step e Gels Thermo Scientific Precise Protein Gels PI25200 and others are cast in a durable plastic cassette with a neutral pH buffer that prevents polyacrylamide breakdown and results in a long shelf life e Gel Electrophoresis Buffers we offer a variety of ready to use pre formulated buffers for protein methods such as Thermo Scientific BupH Tris Hepes SDS Running Buffer PI28398 Lane Marker Reducing Sample Buffer 5X PI39000 Lane Marker Non Reducing Sample Buffer 5X
4. subsidiaries Specifications terms and pricing are subject to change Not all products are available In the United States In Canada ermo Fisher For customer service call 1 800 766 7000 For customer service call 1 800 234 7437 gt cae To fax an order use 1 800 926 1166 To fax an order use 1 800 463 2996 in all countries Please consult your local sales representative for details SCIENTIFIC Scientific To order online www fishersci com To order online www fishersci ca BNO927133 10 13 Printed in the U S Part of Thermo Fisher Scientific
5. a variety of types including nitrocellulose PI88018 PVDF PI88518 and low fluores cence PVDF PI22860 We also offer Western blotting filter paper in regular s PI88600 PI84783 and PI84784 and extra thick PI88605 PI88610 PI88615 and PI88620 sheets ol Q ajs Blocking Next block the unreacted sites on the membrane to reduce the amount of nonspecific binding We have a complete selection of blocking buffers to improve the sensitivity of your Western blot The proper choice of buffer depends on the antigen and type of enzyme conjugate to be used With the wide range we offer choose the one that delivers the highest signal to noise ratio possible for your blots We have a wide selection of Thermo Scientific blocking buffers available to meet the need of just about any Western blot protocol Visit thermoscientific com blockingbuffers for our blocking buffer selection guide e Blocker BLOTTO e StartingBlock Blocking Blocking Buffer Buffers e Pierce Clear Milk Blocking Buffer e Pierce Fast Blocking Buffer e SuperBlock Blocking Buffers e Blocker BSA Blocking Buffers e Blocker Casein Blocking e SEA BLOCK Blocking Buffer Buffers e Protein free Blocking Buffer e Normal Serum Il I l L O S T Primary Incubation Incubate the membrane with primary antibody Our antibodies are fully validated eliminating the need to screen numerous antibodies to find the correct one Visit ther
6. bstrate PI34078 Our most popular substrate can be easily optimized to detect targets with greater sensitivity than ECL substrates Visit www thermoscientific com Western to see our complete line of chemiluminescent and colorimetric substrates Target Detection Capture and analyze your image Select your mode of target detection Choose between traditional detection using X ray film or the more quantitative cooled CCD camera imaging technology We offer both Thermo Scientific CL XPosure Film s PI34089 PI34090 PI34091 is economically priced clear blue film for detection of chemiluminescent Western blots Or take your imaging to the next level with the Thermo Scientific myECL Imager for one touch image capture of Western blots With the Thermo Scientific mylmageAnalysis Software PI62237 the mYECL Imager is a powerful tool to analyze and quantify your target bands The myECL Imager PI62236 is a powerful and easy to use blot and gel documentation instrument for sensitive multimode image capture and analysis via an intuitive touchscreen interface and advanced integrated software The myECL Imager works with chemiluminescent colorimetric or UV light activated fluorescent substrates or stains if you are detecting with X ray film use Thermo Scientific Pierce Background Eliminator Kit PI32065 for fast easy removal of artifacts to correct for overexposure of blots Stripping if necessa
7. e a different blocking buffer e Do not use milk with avidin biotin systems Milk contains biotin e Test for cross reactivity Block a clean piece of membrane incubate with antibodies and then detect with SuperSignal Chemiluminescent Substrate e Reduce the concentration of the HRP conjugate 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 Cover the membrane with liquid at all times to prevent it from drying e Use agitation during all incubations e Incubate membranes separately to ensure that membrane strips are not covering one another during incubations e Handle membranes carefully damage to the membrane can cause nonspecific binding Contamination in buffers e Use new buffers e Filter buffers before use Contaminated equipment e Use only clean and contaminant free electrophoresis equipment blotting equipment and incubation trays e No pieces of gel should be left on the membrane after transfer because proteins can stick to them causing background a Pi Lo i Possible Causes Precautions Solutions Antibody concentrations are too high e 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 A gee g gt x y N i AN
8. ergent PI28320 Surfact Amps Detergents Packs PI28372 e Tween 80 Detergent P128328 e 20X Phosphate Buffered Saline e Triton X 100 Detergent PI28348 PI28314 NP 40 PI28324 e Tris Buffered Saline s PI28376 PI28358 A jf a 2 P l S y mo E Hi 2 4am ee diy Neem E smo Incubation with Substrate Add the detection reagent to your secondary HRP or AP conjugate Choose the appropriate substrate for your needs from the Thermo Scientific Pierce ECL and Thermo Scientific SuperSignal families of chemiluminescent HRP substrates All of our substrates offer excellent performance in Western blotting with long light emission and strong signal intensity We offer five types of chemiluminescent substrates for Western blot detection with HRP e Pierce ECL Substrate PI32106 SuperSignal West Dura Extended An entry level substrate with Duration Substrate PI34076 low picogram level sensitivity Offers high sensitivity and Select when the sample target 24 hour signal output that is ideal is abundant for CCD camera and other digital e Pierce ECL Plus Substrate Imaging systems PI32132 Detect down to 0 5pg SuperSignal West Femto of your target Select when Maximum Sensitivity Substrate target is less abundant and PI34096 Our most sensitive sample is limited substrate Select when sample is limited and or target is less e SuperSignal West Pico abundant Chemiluminescent Su
9. fied Dubecco s PBS Buffer 8 PI28344 PI28374 Skip this step if you use Thermo Scientific StartingBlock T20 Blocking Buffer in PBS or TBS or Thermo Scientific SuperBlock T20 Blocking Buffer in PBS or TBS These buffers already contain Tween 20 Detergent at optimized concentrations gt ws WANAR k h For technical informati n video tutorials and selection guides visit thermoscientific com Western Secondary Incubation Incubate the membrane with secondary antibody Choose an appropriate secondary detection probe for your Western blot Our secondary antibodies and detection reagents are available in a variety of formats and conjugated types including HRP AP DyLight Dyes and others Check out our secondary antibody selection guide to find the secondary antibody or detection reagent that is right for you We also offer a complete line of biotin binding proteins and conjugates Avidin Streptavidin etc antibody binding proteins Protein A Protein G etc and specialized detection probes and kits Visit thermoscientific com Western for a complete list thermoscientific com pierce abs Wash Remove unbound secondary reagents and reduce background Our dry buffers and high purity detergents all serve to enhance your signal to noise ratio Powerful easy to use solutions help achieve the cleanest results possible Buffered Saline Solutions e BupH Phosphate Buffered Saline Tween 20 Det
10. instructions e Use new membranes e Cover the membrane with liquid at all times to prevent it from drying Insufficient washing 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 e Prepare new buffers in buffers Possible Causes Precautions Solutions Antibody concentrations e High concentrations of primary and or secondary antibody can cause high background are too high e Decrease antibody concentrations Aggregate formation in the HRP e Filter the conjugate through a 0 2um filter conjugate can cause speckling e Use a new high quality conjugate Incompatible blocking buffer e Compare different blocking buffers was used Insufficient blocking e Optimize blocking buffer The best blocking buffer is system dependent of 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 Detergent to the blocking buffer to a final concentration of 0 05 Skip this step if you use StartingBlock T20 Blocking Buffer in PBS or TBS or SuperBlock T20 Blocking Buffer in PBS or TBS e Make up antibody dilutions in blocking buffer with 0 05 Tween 20 Detergent Cross reactivity of antibody with other proteins in blocking buffer e Us
11. moscientific com pierce abs to find your antibody Thermo Scientific Pierce Antibodies are developed for a wide variety of application needs We offer over 40 000 antibodies for over 50 research areas and all of our antibodies are validated and guaranteed to perform in the stated application and species We also offer antibody conjugates available with Thermo Scientific DyLight Dyes biotin horseradish peroxidase HRP alkaline phosphatase AP and more Our website enables you to easily search by protein target and then filter by the specific assays that interest you Our custom antibody service leverages our experience and proprietary antigen design tools to produce more robust antibodies 1 i thermoscientific com pierce abs O A i pa a lt 4 E aes a kn Sh 4 Ao k wk i a 4 Wash Remove unbound primary reagents and reduce background Our dry blend buffers and high purity detergents all serve to enhance your signal to noise ratio Powerful easy to use Thermo Scientific solutions help achieve the cleanest results possible Buffered Saline Solutions Thermo Scientific e BupH Phosphate Buffered Surfact Amps Detergents Saline Packs PI28372 e Tween 20 Detergent PI28320 e Pierce 20X Phosphate Buffered Tween 80 Detergent PI28328 saline PI26348 e Triton X 100 Detergent e BupH Tris Buffered Saline PI28314 NP 40 PI28324 s PI28376 PI28358 e Modi
12. ot to determine activity Antibody concentrations e Using too much primary or secondary antibodies can cause the signal to fade quickly are too high Insufficient amount of e Load more protein onto the gel antigen present The antigen is masked o Try different blocking buffers by the blocking buffer e Optimize blocking buffer protein concentration Buffers contain sodium azide e Do not use sodium azide an inhibitor of HRP as a preservative in buffers Exposure time is too short e Lengthen the film exposure time Substrate incubation e SuperSignal Substrates require a 5 minute substrate incubation is too short Inactive substrate e Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate and Thermo Scientific SuperSignal West Dura Chemiluminescent Substrate are stable for up to 12 months at RT Thermo Scientific SuperSignal West Femto Chemiluminescent Substrate is stable for at least six months at RT e To evaluate the substrate activity prepare a small amount of working solution In a darkroom add a small amount of HRP conjugate A blue light should be observed If no glow is observed either the substrate or the HRP conjugate is inactive e Ensure there is no cross contamination between the two bottles of substrate which can cause a decline in activity Membrane has been stripped and reprobed e Optimize stripping procedure to prevent any loss of antigen or denaturation e Reprobe only when nece
13. ry Reprobe the blot if needed Using our Thermo Scientific Restore Products you can quickly strip and reprobe as well as reuse the blot again and again Strip time off your research processes Reprobing a Western blot saves time and conserves sample while allowing optimization to be performed as needed Reprobing also allows the same blot to be probed for different target proteins We offer specially formulated buffers that are developed to efficiently strip primary and secondary antibodies from Western blots so that membranes can be reprobed under alternate conditions or with another antibody to detect a different protein target Thermo Scientific Stripping Buffers e Restore Western Blot Stripping Buffer PI21059 e Restore PLUS Western Blot Stripping Buffer Pl46430 e Restore Fluorescent Western Blot Stripping Buffer PI62300 IO MC see more learn more Possible Causes Precautions Solutions Antibody concentrations e High concentrations of primary and or secondary antibody can cause high background are too high e Decrease antibody concentrations Incompatible blocking buffer was used e Optimize blocking buffer The best blocking buffer is system dependent 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 0 05 Tween 20 Detergent to the blocking buffer at a final concen
14. ssary e Avoid repeated reprobing of the same membrane Digestion of antigen e Blocking substance may have proteolytic activity e g gelatin on the membrane Protein degradation e Prepare a new blot from blot storage Possible Causes Precautions Solutions Antibody concentrations e Reduce antibody concentrations are too high 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 e Reduce antibody concentrations are too high Too much protein is loaded onto the gel e Reduce the amount of protein loaded onto the gel Possible Causes Precautions Solutions Incomplete transfer of proteins e Make sure there are no air bubbles between the gel and membrane during transfer from the gel e Wet membrane according to the manufacturer s instructions e Do not handle the membrane with bare hands Always wear clean gloves or use forceps e Use a new membrane e Incubate membranes separately to ensure that membrane strips are not covering one another during incubations thermoscientific com pierce 2013 Thermo Fisher Scientific Inc All rights reserved These products are supplied for laboratory or manufacturing applications only Tween is a trademark of ICI Americas Triton is a trademark of Rohm and Haas Company All other trademarks are the property of Thermo Fisher Scientific Inc and its
15. tration of 0 05 This is not applicable to Thermo Scientific StartingBlock T20 Blocking Buffer in PBS or TBS or Thermo Scientific SuperBlock T20 Blocking Buffer in PBS or TBS e Prepare antibody dilutions in blocking buffer that contains 0 05 Tween 20 Detergent Hang this poster in your lab to help you avoid or overcome problems in your Western Cross reactivity of antibody with other proteins in blocking buffer e Use a different blocking buffer Thermo Scientific Pierce Protein free Blocking Buffers are PBS and TBS formulations of a non protein compound for effective membrane and plate blocking with extremely low background e Do not use milk with avidin biotin systems Milk contains biotin e Test for cross reactivity Block a clean piece of membrane incubate with antibodies and then detect with Thermo Scientific SuperSignal Chemiluminescent Substrate e Reduce the concentration of the HRP conjugate blotting application e Increase number of washes and the volume of buffer used e Add Tween 20 Detergent to wash buffer at a final concentration of 0 05 Note 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 or TBS or SuperBlock T20 Blocking Buffer in PBS or TBS Exposure time is too long e Reduce the time the blot is exposed to film Membrane problems e Wet membranes thoroughly according to the manufacturer s
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