Introduction to Western Blotting

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Protein transfer from gel to membrane - Western Blotting

Overview

After separation by electrophoresis, proteins can be bound to membranes where they are fixed and readily accessible for immunological or biochemical analyses, quantitative staining, or the identification of protein-protein and protein-ligand interactions. Western blotting, the transfer of proteins to a solid-phase membrane support followed by immunodetection, is a powerful and popular technique for the visualization and identification of proteins. Western blotting combines the resolution of gel electrophoresis with the specificity of immunoassays, allowing individual proteins in mixtures to be identified and analyzed. This section provides an overview of western blotting methods, equipment, membranes, transfer buffers, transfer conditions, detection, and imaging.

A special section, the Western Blot Doctor™, is a self-help guide developed by Bio-Rad researchers that enables you to identify and troubleshoot western blotting problems. Comprehensive solutions and suggestions are provided to help solve your particular western blotting challenges.

 

See how you can streamline your protein electrophoresis and western blotting experiments.

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Stain-Free Technology
 
 
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Western Blotting Workflow

The most commonly used protein blotting technique, western blotting (immunoblotting), was developed to probe for proteins that were inaccessible to antibodies while in polyacrylamide gels. Western blotting refers specifically to the immunological detection of proteins that have been separated by gel electrophoresis and transferred onto a membrane. Since the development of immunoblotting techniques, other probing and detection techniques have been developed for functional protein characterization (for a review, see Kurien and Scofield 2003).

The western blotting workflow involves two phases: protein transfer to a membrane and detection of the membrane-immobilized protein.

Protein Transfer to a Membrane
The first phase of western blotting is the transfer step, which consists of moving the proteins from a solution or gel matrix to a synthetic membrane support where it is bound, forming the blot. Proteins can be transferred to membranes using a number of methods, but the most common are electrophoretic transfer (electroblotting) and microfiltration (dot blotting). In general, electrophoretic transfer is used to transfer proteins following electrophoretic separation by native or SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and microfiltration is used to transfer proteins that are in solution. Capillary blotting methods, such as those used for nucleic acid transfer, are seldom used to transfer proteins from gels.

Protein Detection on the Membrane
The second phase, protein detection, entails probing the membrane with either a protein stain or antibodies specific to the protein of interest, and subsequent visualization of the labeled proteins. Western blot detection involves a number of steps, including selection of the appropriate protein detection method, blotting buffers and reagents, and gel and blot imaging equipment.

The protein blotting workflow involves selection of the appropriate method, apparatus, membrane, buffer, and transfer conditions. Once proteins are immobilized on a membrane, they are available for visualization, detection, and analysis.

Diagram of western blotting workflow - western blotting

Select the method Select the equipment Prepare the reagent Perform the transfer

 

Further Reading

Kurien BT and Scofield RH (2003). Protein blotting: a review. J Immunol Methods 274, 1–15.

Jensen CE (2012). The basics of western blotting. Anatomical Record 295(3) 369–371.

 

Related Content

 
Literature
Number Description Download
6570 Experimental Protocol for Multiplex Fluorescent Blotting Using the ChemiDoc™ MP Imaging System, Rev A Click to download
2032 Western Blotting Detection Reagents Brochure, Rev F Click to download
2895 Protein Blotting Guide, Ver C Click to download
6040 Electrophoresis Guide, Interactive PDF, Rev C Click to download
6350 Housekeeping Protein Normalization Publications, Rev A Click to download
6351 Selected Publications List: Total Protein Normalization in Western Blotting Using Stain-Free Technology Click to download
6356 Traditional vs. V3 Western Workflow, Ver B Click to download
6390 General V3 Western Workflow Blotting Protocol Click to download
RP0058 A Defined Methodology for Reliable Quantification of Western Blot Data Click to download
6359 Avoiding Housekeeping Protein Detection Saturation Protocol, Rev A Click to download
6361 Determining the Appropriate Film Exposure Time Protocol, Rev A Click to download
6362 Determining the Appropriate Sample Load for Western Blots Protocol, Rev A Click to download
6363 Determining the Appropriate Sample Load When Using a Stain-Free V3 Western Workflow™ Protocol, Rev A Click to download
6366 Validating the Expression Consistency of a Housekeeping Protein Protocol, Rev A Click to download
6376 General Protocol for Western Blotting Protocol, Rev A Click to download
6390 General V3 Western Workflow Blotting Protocol, Rev A Click to download
 
 
LUSPPAKG4 [x-forwarded-proto] = [http] [x-forwarded-port] = [80] [x-forwarded-for] = [54.167.80.159, 10.232.18.13] [accept] = [*/*] [seourl] = [/en-de/applications-technologies/2-d-electrophoresis] [x-amzn-trace-id] = [Root=1-5d0618d1-e3833b2fc837dc152568ff25] [x-forwarded-server] = [lsds-prod-s.br.aws-livesite.io] [x-forwarded-host] = [www.bio-rad.com] [x-query-string] = [ID=LUSPPAKG4] [host] = [10.232.17.28:1776] [x-request-uri] = [/en-de/applications-technologies/2-d-electrophoresis] [connection] = [Keep-Alive] [accept-encoding] = [gzip, deflate] [user-agent] = [rogerbot/1.2 (https://moz.com/help/guides/moz-procedures/what-is-rogerbot, rogerbot-crawler+aardwolf-production-crawler-53@moz.com)] AppTech/AppTechDetails pageStyleKey internet/solutions_sub applications-technologies/2-d-electrophoresis LSR LUSPPAKG4 Western Blotting Introduction to Western Blotting /webroot/web/html/lsr/solutions/technologies/western_blotting /webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/technology_detail/western-blotting-feature.jpg /webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/technology_thumb/western-blotting-thumb.jpg Protein transfer from gel to membrane - Western Blotting <script type="text/javascript">// <![CDATA[ if ($.browser.msie && $.browser.version < 8) {$("div.methodboxmiddle ul.rightarrowsearch1").css({"margin-left":"-5px"});} // ]]></script> <p>After separation by electrophoresis, proteins can be bound to membranes where they are fixed and readily accessible for immunological or biochemical analyses, quantitative staining, or the identification of protein-protein and protein-ligand interactions. Western blotting, the transfer of proteins to a solid-phase membrane support followed by immunodetection, is a powerful and popular technique for the visualization and identification of proteins. Western blotting combines the resolution of gel electrophoresis with the specificity of immunoassays, allowing individual proteins in mixtures to be identified and analyzed. This section provides an overview of western blotting methods, equipment, membranes, transfer buffers, transfer conditions, detection, and imaging.</p> <p><!-- <a href="/evportal/destination/solutions?catID=MIW4X7MNI" mce_href="/evportal/destination/solutions?catID=MIW4X7MNI">Western Blotting protocols</a> and tips are also provided. -->A special section, the <a href="/evportal/destination/solutions?catID=MIW4HR15">Western Blot Doctor&trade;</a>, is a self-help guide developed by Bio-Rad researchers that enables you to identify and troubleshoot western blotting problems. Comprehensive solutions and suggestions are provided to help solve your particular western blotting challenges.</p> <p>&nbsp;</p> <div class="bannerAT"> <div class="bannerText ddpcrText" style="width: 390px !important;"> <h2 class="banner_header">Stain-Free Technology</h2> <p style="padding: 0 !important; margin: 10px 0 !important;">See how you can streamline your protein electrophoresis and western blotting experiments.</p> <a class="linkgeneration" href="/en-us/applications-technologies/stain-free-technology">Find out more &raquo;</a></div> <img src="/webroot/web/images/lsr/global/english/solutions/stain-free-technology.jpg" alt="Stain-Free Technology" width="615" height="120" /></div> <script src="/webroot/web/js/countrySpecific-min.js" type="text/javascript"></script> <script type="text/javascript">// <![CDATA[ $(document).ready(function(){ setSterlingUrlsToHtmlHrefVariables(); $('a.linkgeneration').each(function(){ $(this).attr('href', $(this).attr('href').replace('_locale', languageCode + '-' + countryCode).replace('_verticalUrl', currentVerticalUrlTitle).replace('_defaultVerticalUrl', defaultVerticalUrlTitle).replace('_feedbackCMSID',feedbackCMSID)); }); }); // ]]></script> Western Blotting Workflow <p>The most commonly used protein blotting technique, western blotting (immunoblotting), was developed to probe for proteins that were inaccessible to antibodies while in polyacrylamide gels. Western blotting refers specifically to the immunological detection of proteins that have been separated by gel electrophoresis and transferred onto a membrane. Since the development of immunoblotting techniques, other probing and detection techniques have been developed for functional protein characterization (for a review, see <a href="#kurien">Kurien and Scofield 2003</a>).</p> <p>The western blotting workflow involves two phases: protein transfer to a membrane and detection of the membrane-immobilized protein.</p> <p><strong>Protein Transfer to a Membrane</strong><br /> The first phase of western blotting is the transfer step, which consists of moving the proteins from a solution or gel matrix to a synthetic membrane support where it is bound, forming the blot. Proteins can be transferred to membranes using a number of methods, but the most common are electrophoretic transfer (electroblotting) and microfiltration (dot blotting). In general, electrophoretic transfer is used to transfer proteins following electrophoretic separation by native or <a href="/evportal/destination/solutions?catID=LUSOVO47B">SDS-polyacrylamide gel electrophoresis</a> (SDS-PAGE), and microfiltration is used to transfer proteins that are in solution. Capillary blotting methods, such as those used for nucleic acid transfer, are seldom used to transfer proteins from gels.</p> <p><strong>Protein Detection on the Membrane</strong><br /> The second phase, protein detection, entails probing the membrane with either a protein stain or antibodies specific to the protein of interest, and subsequent visualization of the labeled proteins. Western blot detection involves a number of steps, including selection of the appropriate <a href="/evportal/destination/solutions?catID=LUSPULKSY">protein detection method</a>, <a href="/evportal/destination/solutions?catID=LUSQA88UU">blotting buffers and reagents</a>, and <a href="/evportal/destination/solutions?catID=LUSQCPKSY">gel and blot imaging equipment</a>.</p> <p>The protein blotting workflow involves selection of the appropriate method, apparatus, membrane, buffer, and transfer conditions. Once proteins are immobilized on a membrane, they are available for visualization, detection, and analysis.</p> <p><a name="workflowimg"></a></p> <p><img usemap="#wfmap" src="/webroot/web/images/lsr/solutions/technologies/protein_electrophoresis_blotting_and_imaging/western_blotting/technology_detail/western-blotting-workflow-western-blotting.jpg" border="0" alt="Diagram of western blotting workflow - western blotting" width="530px" height="590px" /></p> <p><map name="wfmap"> <area shape="rect" coords="51,59,233,85" href="/evportal/destination/solutions?catID=LUSPPSESH" alt="Select the method" /> <area shape="rect" coords="48,175,230,197" href="/evportal/destination/solutions?catID=LUSPQXC4S" alt="Select the equipment" /> <area shape="rect" coords="52,279,232,301" href="/evportal/destination/solutions?catID=LUSPSJ8UU" alt="Prepare the reagent" /> <area shape="rect" coords="51,402,228,425" href="/evportal/destination/solutions?catID=LUSPTIMNI" alt="Perform the transfer" /> </map></p> <div class="top"><a href="#helptop">Back to Top</a></div> Further Reading <p><a name="kurien"></a>Kurien BT and Scofield RH (2003). Protein blotting: a review. J Immunol Methods 274, 1&ndash;15.</p> <p>Jensen CE (2012). The basics of western blotting. Anatomical Record 295(3) 369&ndash;371.</p> <div class="top"><a href="#helptop">Back to Top</a></div> Protocols <table id="carttablealigned" class="literature_table" style="height: auto; width: 583px;" border="0" cellspacing="0" cellpadding="0"> <tbody> <tr> <td width="100">6359</td> <td width="350">Avoiding Housekeeping Protein Detection Saturation Protocol, Rev A</td> <td class="pdf"><a class="pdf" href="/webroot/web/pdf/lsr/literature/Bulletin_6359.pdf" target="_blank" rel="noopener noreferrer"><span>Click to download</span></a></td> </tr> <tr> <td>6361</td> <td>Determining the Appropriate Film Exposure Time Protocol, Rev A</td> <td class="pdf"><a class="pdf" href="/webroot/web/pdf/lsr/literature/Bulletin_6361.pdf" target="_blank" rel="noopener noreferrer"><span>Click to download</span></a></td> </tr> <tr> <td width="100">6362</td> <td width="350">Determining the Appropriate Sample Load for Western Blots Protocol, Rev A</td> <td class="pdf"><a class="pdf" href="/webroot/web/pdf/lsr/literature/Bulletin_6362.pdf" target="_blank" rel="noopener noreferrer"><span>Click to download</span></a></td> </tr> <tr> <td>6363</td> <td>Determining the Appropriate Sample Load When Using a Stain-Free V3 Western Workflow&trade; Protocol, Rev A</td> <td class="pdf"><a class="pdf" href="/webroot/web/pdf/lsr/literature/Bulletin_6363.pdf" target="_blank" rel="noopener noreferrer"><span>Click to download</span></a></td> </tr> <tr> <td>6366</td> <td>Validating the Expression Consistency of a Housekeeping Protein Protocol, Rev A</td> <td class="pdf"><a class="pdf" href="/webroot/web/pdf/lsr/literature/Bulletin_6366.pdf" target="_blank" rel="noopener noreferrer"><span>Click to download</span></a></td> </tr> <tr> <td>6376</td> <td>General Protocol for Western Blotting Protocol, Rev A</td> <td class="pdf"><a class="pdf" href="/webroot/web/pdf/lsr/literature/Bulletin_6376.pdf" target="_blank" rel="noopener noreferrer"><span>Click to download</span></a></td> </tr> <tr> <td>6390</td> <td>General V3 Western Workflow Blotting Protocol, Rev A</td> <td class="pdf"><a class="pdf" href="/webroot/web/pdf/lsr/literature/Bulletin_6390.pdf" target="_blank" rel="noopener noreferrer"><span>Click to download</span></a></td> </tr> </tbody> </table> 6570 /templatedata/internet/documentation/data/LSR/Literature/6570_1407868598.xml 2032 2895 6040 /templatedata/internet/documentation/data/LSR/Literature/6040.xml 6349 /templatedata/internet/documentation/data/LSR/Literature/6349.xml 6350 /templatedata/internet/documentation/data/LSR/Literature/6350.xml 6351 /templatedata/internet/documentation/data/LSR/Literature/6351.xml 6356 /templatedata/internet/documentation/data/LSR/Literature/6356.xml 6390 /templatedata/internet/documentation/data/LSR/Literature/6390.xml RP0058 /templatedata/internet/documentation/data/LSR/Literature/RP0058_1381251352.xml Life Science Research/Products/Electrophoresis and Blotting/Protein Electrophoresis and Blotting/Mini Format 1D-Electrophoresis Systems/Mini-PROTEAN Tetra Cell Systems ->MT::5cf78e19-7ed5-4373-a988-3e62456a488e##Life Science Research/Products/Electrophoresis and Blotting/Protein Electrophoresis and Blotting/Mini Format 1D-Electrophoresis Systems/Mini-PROTEAN Precast Gels/Mini-PROTEAN TGX Precast Gels ->MTS::KSHTFUE8Z##Life Science Research/Products/Electrophoresis and Blotting/Protein Electrophoresis and Blotting/Western Blotting/Semi-Dry Blotting Systems/Trans-Blot Turbo Transfer System ->MTS::LGOQBW15##Life Science Research/Products/Electrophoresis and Blotting/Protein Electrophoresis and Blotting/Western Blotting/Wet/Tank Blotting Systems/Mini Trans-Blot Cell ->MT::589ca8f7-5751-487a-a453-571ee8cc8b7e##Life Science Research/Products/Imaging Instruments &amp; Bioinformatics/Molecular Imager Systems/Gel Doc EZ Systems ->MTS::L7BL4S15## Life Science Research/Solutions/Technologies/Protein Electrophoresis ->MTS::LUSOVO47B##Life Science Research/Solutions/Technologies/Imaging and Analysis ->MTS::LUSQC6MNI##Life Science Research/Solutions/Applications/Protein Separation and Analysis/Fast Separation Transfer and Analysis ->MTS::LUSOUMFCN## Eddie C Introduction to Western Blotting Learn more about western blotting techniques. Find step-by-step protocols and helpful tips on equipment, membranes, transfer conditions, and detection methods. western blotting, protein separation, protein transfer, blocking nonspecific sites, wash buffer formulation, primary antibody, secondary antibody, detection, immunodetection, biochemical analysis, western blot doctor, stain-free technology, protein blotting, housekeeping protein normalization, protein quantification, 12/08/11 10:33 AM 12/08/21 10:37 AM AE,AI,AL,AM,AR,AT,AU,AZ,BA,BD,BE,BF,BG,BH,BN,BO,BR,BW,CA,CH,CL,CM,CN,CO,CR,CY,CZ,DE,DK,DO,DZ,EC,EE,EG,EH,ER,ES,ET,FI,FM,FO,FR,GA,GE,GF,GH,GP,GR,GT,GU,HK,HN,HR,HT,HU,ID,IE,IL,IN,IS,IT,JM,JO,JP,KE,KH,KR,KW,KZ,LB,LI,LK,LT,LU,LV,MA,MD,MG,MK,ML,MO,MQ,MS,MT,MU,MX,MY,NG,NI,NL,NO,NP,NZ,OM,PA,PE,PF,PG,PH,PK,PL,PR,PS,PT,PW,PY,QA,RO,RS,RU,SA,SB,SE,SG,SI,SK,SN,ST,SV,TG,TH,TN,TO,TR,TT,TW,TZ,UA,UG,UK,US,UY,UZ,VA,VE,VU,XK,YE,ZA,VN en LSR /LSR/Technologies/Western_Blotting N 0
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