CINXE.COM

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5.1724696046-473375462.1712005244" style="margin: 0 10px;" target="_blank"><img alt="Novus Biologicals products are now on bio-techne.com" src="https://images.novusbio.com/design/ready-to-buy-novus-products-login.png" style="height:120px; width:940px" /></a></div> </div> </div> </div> <h1>Western Blotting</h1> </div> <div class="clear"></div> <div class="grid_16"> <div class="region region-content"> <div id="block-et-messages-et-messages-block" class="block block-et-messages"> <div class="content"> <span></span> </div> </div> <div id="block-system-main" class="block block-system"> <div class="content"> <div id="node-18144671" class="node node-page clearfix"> <div class="content"> <div class="field field-name-body field-type-text-with-summary field-label-hidden"><div class="field-items"><div class="field-item even"><table border="0" cellpadding="6" cellspacing="6"> <tr> <td colspan="2"> <img src="//images.novusbio.com/design/westernBlottingHeader03.png" alt="Western Blotting Products and Resources" > </td> </tr> <tr> <td width="208" valign="top" style="width:220px;"> <p><b><u>Related Links</u></b></p> <p><a href="https://www.bio-techne.com/resources/literature/western-blot-handbook" target="_blank">Western Blot Handbook</a></p> <p><a href="/support/support-by-application/western-blot/protocol.html">Western Blot Protocol</a></p> <p><a href="/support/western-blot-resources">Western Blot Protocols, Troubleshooting, & Scientific Resources</a></p> <p><a href="/support/SecondaryAntibodyHandbook">Secondary Antibody Handbook</a></p> <p ><a href="/search?keywords=novus&category=Primary%20Antibodies&applications=Western%20Blot">Validated Antibodies for Western Blot</a></p> <p><a href="/product-type/primary-antibodies?applications=Simple%20Western">Simple Western Antibodies </a></p> <p><a href="/support/simple-western-faqs">Simple Western FAQs</a></p> <p><a href="/application/single-cell-western">Single-Cell Western </a></p> <p><a href="/support/single-cell-western-faqs">Single-Cell Western FAQs </a></p> <p><a href="/category/western-blot?page=1">Western Blot Blogs</a></p> </td> <td width="400"> <p>Western blotting is the gold standard for identification and quantification of proteins from a lysate, providing averaged protein expression from thousands of pooled cells. This technique can also be used to determine sub-cellular localization (via <a href="/support/cell-fractionation-and-organelle-isolation">cell fractionation</a>), post-translational modifications (e.g. ubiquitination, phosphorylation), protein processing, and protein-protein interactions, when coupled with <a href="/support/support-by-application/immunoprecipitation/protocol.html">immunoprecipitation</a>. When sample volume is limited or there is a large workload, the fully automated <a href="https://www.bio-techne.com/instruments/simple-western" target="blank">Simple Western™</a> platform is a hands-off approach to achieve <a href="https://www.bio-techne.com/simple-western/automated-simple-western-blot-analysis-cost" target="blank">reproducible results in less time</a>.</p> <br/> <p align="center"><a href="/support/support-by-application/western-blot/protocol.html" class="link_button">View Western Blot Protocol</a></p> <br/> <p align="center"> <img src="//images.novusbio.com/design/wb-application-illustration.jpg" alt="Conventional western blot application allows visualization of proteins through separation, transfer and antibody staining steps using Novus antibodies." title="Principles of Western Blot" > </p> <p style="margin-left:36px; margin-right:30px;"> <em><strong>Overview of Conventional Western Blot Protocol</strong>. (A) Proteins are separated by polyacrylamide gel electrophoresis (PAGE) and (B) transferred to a membrane (e.g. nitrocellulose or PVDF) for detection. (C) The membrane is probed with a primary antibody specific for the target protein and typically followed by an enzyme conjugated secondary antibody to detect the antibody-antigen complex. The enzyme (e.g. horseradish peroxidase, HRP) acts on a substrate (e.g. electro-chemiluminescence, ECL) to emit light, (D) generating a signal captured on autoradiography film or a chemiluminescence imaging system. <a href="/support/support-by-application/western-blot/protocol.html">Learn more about perfecting Western blots</a>.</em> </p> <br/> <div><a href="https://www.bio-techne.com/resources/literature/western-blot-handbook " target="_blank"><img src="//images.novusbio.com/design/novus-wblot-banner.png" alt="western blot handbook" width="700"></a></div> <br/> <br/><br/> <h2>Western Blot Primary Antibodies</h2> <p> The success of a western blot is often dependent upon the specificity of the primary antibody. Novus Biologicals employs the <a href="/5-pillars-validation">5 Pillars of Validation</a> to verify antibody specificity, including genetic validation by knockout (KO) or knockdown (KD) strategies. Novus offers a broad selection of highly rated monoclonal and recombinant primary antibodies backed <a href="/support/quality-assurance.html">by our 100% guarantee</a> and can be tested in unvalidated species with our <a href="/support/innovators-reward.html">Risk-Free Testing program.</a> <a href="/products/primary-antibodies">Learn more about primary antibodies</a>. </p> <p>The <a href="https://www.bio-techne.com/resources/simple-western-antibody-database" target="_blank">Simple Western Antibody Database</a> on Bio-Techne.com is a user-interactive listing of all Simple Western validated antibodies to date. <a href="https://www.bio-techne.com/promotions/simple-western-antibody-validation-submission" target="_blank">Submit your antibody validation data</a> and get a free Separation or Detection Module!</p> <table> <tr> <td colspan="2" valign="top"> <b>Choose from:</b> </td> </tr> <tr> <td valign="top"> <ul> <li><a href="/primary-antibodies/blue-marker">Blue Marker Antibody</a></li> <li><a href="/search?keywords=novus&category=Primary%20Antibodies&applications=Knockout%20Validated">Primary Antibodies - KO Validated</a></li> <li><a href="/product-type/primary-antibodies?research_areas=Loading%20Controls">Primary Antibodies - Loading Controls</a></li> <li><a href="/search?keywords=%22recombinant%20monoclonal%20antibodies%22&category=Primary%20Antibodies">Primary Antibodies – Recombinant Monoclonal</a></li> <li><a href="/search?keywords=epitope%20tags&category=Primary%20Antibodies">Primary Antibodies - Epitope Tag</a></li> </ul> </td> <td valign="top"> <ul> <li><a href="/search?keywords=novus&facet_conjugate=HRP">Primary Antibodies – HRP</a></li> <li><a href="/search?keywords=novus&facet_conjugate=Alkaline%20Phosphatase">Primary Antibodies - AP</a></li> <li><a href="/search?keywords=novus&category=Primary%20Antibodies&applications=Simple%20Western">Primary Antibodies – Simple Western Validated</a></li> <li><a href="/search?keywords=novus&category=Primary%20Antibodies&applications=Western%20Blot">Primary Antibodies - Western Blot Validated</a></li> <li><a href="/research-areas/cellular-markers">Primary Antibodies - Organelle Markers</a></li> <li><a href="/Custom Conjugation Services">Custom Conjugation Service</a></li> </ul> </td> <tr> </table> <br/><br/> <p align="center"> <a href="https://www.bio-techne.com/p/simple-western/jess_004-650" target="blank"><img src=" https://images.novusbio.com/design/protein-simple-jess-instrument-ad-v4.jpg" alt="Simple Western application saves time and allows users to have high throughput western data using automated western blot machines from Protein Simple. " title="Learn about fully automated westerns by using Simple Western." border="0" width="700" ></a> </p> <table align="center" border="0" cellpadding="2" class="top"> <tbody> <tr> <td colspan="2"><h4>Accelerate your assay development on Simple Western - Join our live online workshops!</h4></td></tr><tr> <td><a href="https://academy.bio-techne.com" target="_blank"><img alt="Bio-Techne Academy" src="https://resources.bio-techne.com/bio-techne-assets/images/support/bio-techne-academy-logo.png" width="250" height="112.5"></a></td><td><p>Join our live and online workshops to learn from our scientists to optimize assays across the development pipeline, accurately analyze data using Compass software, and more. Join us in the Bio-Techne Academy, starting on <b>January 26th, 2023!</b></p> <p><a class="link_button" href="https://academy.bio-techne.com" target="_blank" title="Register for live workshops">Register for Live Workshops</a></p> </td></tr> </tbody> </table> <br/><br/> <h2>Secondary Antibodies and Detection Reagents</h2> <p> The detection of primary antibody-antigen complexes on a blot generally requires the use of secondary antibodies. The most common conjugate for secondary antibodies, HRP, is typically used with the chemiluminescent substrate, ECL, to produce an intense signal with low background. While chemiluminescence offers sensitive detection (low picogram) of antigens, fluorescence based detection methods have improved signal stability and allow for simultaneous detection of multiple proteins. <a href="/support/secondary-antibody-handbook">Learn more about secondary antibodies</a>. </p> <table width="709"> <tr> <td colspan="2" valign="top"> <b>Choose from:</b> </td> </tr> <tr> <td width="338" valign="top"> <ul> <li><a href="/search?keywords=novus&category=Secondary%20Antibodies&facet_conjugate=HRP">Secondary Antibodies – HRP</a></li> <li><a href="/search?keywords=novus&category=Secondary%20Antibodies&facet_conjugate=Alkaline%20Phosphatase">Secondary Antibodies - AP</a></li> </ul> </td> <td width="350" valign="top"> <ul> <li><a href="/support/GeneralSecondaryAntibodies">Find Your Secondary Antibody</a></li> <li><a href="/search?keywords=substrate&applications=Western%20Blot&category=Support%20Products">Detection Reagents - HRP and AP</a></li> </ul> </td> </tr> </table> <br/><br/> <h2>Western Blot Controls</h2> <p> Controls are vital for Western blot analysis to validate the specificity of protein bands and/or to uncover the root cause of any issues. Each experiment should include <a href="/support/loading-controls-for-western-blot">loading controls</a> to ensure samples were equally loaded in the gel and to verify the integrity of the sample. Experiments may also require positive control lysates containing the endogenous or overexpressed protein of interest, <a href="/products/crispr-cas9-genetic-knockout-antibody-validation-reproducibility">negative control lysates </a>(knockdown/knockout samples or lysates lacking the expressed protein), or <a href="/support/blocking-with-immunizing-peptide">peptide/protein blocking controls</a> to verify that the band(s) representing the protein of interest is(are) present. </p> <table width="100%"> <tr> <td colspan="2" valign="top"> <b>Choose from:</b> </td> </tr> <tr> <td width="50%" valign="top"> <ul> <li><a href="/product-type/peptides-and-proteins?applications=Blocking/Neutralizing">Blocking Peptides and Proteins</a></li> <li><a href="/product-type/lysates">Control Lysates</a></li> <li><a href="/product-type/lysates?lysate_type=Overexpression">Over-expression Lysates</a></li> </ul> </td> <td width="50%" valign="top"> <ul> <li><a href="/product-type/lysates?lysate_type=Tissue">Tissue Lysates</a></li> <li><a href="/search?keywords=exosome%20standards&applications=Western%20Blot">Exosome Standards</a></li> </ul> </td> </tr> </table> <br/><br/> <p align="center"> <a href="https://www.bio-techne.com/research-areas/targeted-protein-degradation" target="_blank"><img src="//images.novusbio.com/design/protein-degradation-tocris-ad-v5.jpg" alt="Learn about products and services for Targeted Protein Degradation" title="Novus provides INSTA-blot membranes for antibody validation" border="0"></a> </p> <br/> <h2>Other Western Blotting Support Products</h2> <p> In addition to antibodies and detection kits, Novus also offers an assortment of support products for Western blot experiments including an HRP stabilizer, sub-cellular fractionation kits, and Western blot membranes. </p> <table> <tr> <td colspan="2" valign="top"> <b>Choose from:</b> </td> </tr> <tr> <td valign="top"> <ul> <li><a href="/support-products/hrp-conjugate-stabilizer-diluent">HRP Stabilizer</a></li> <li><a href="/search?keywords=novus&category=Inhibitors">Inhibitors</a> & <a href="/product-type/support-products?category=Support%20Products&applications=Ligand%20Activation">Activators (Small Molecules)</a></li> <li><a href="/product-type/support-products?common_name=PBS%20Tablets">PBS Buffer Tablets</a></li> <li><a href="/support/cell-fractionation-and-organelle-isolation">Sub-cellular Fractionation Kits</a></li> </ul> </td> <td valign="top"> <ul> <li><a href="/search?keywords=INSTA%20Blot&category=WB%20Membranes">Western Blot Membranes</a></li> <li><a href="/products/runblue-prestained-molecular-weight-marker-tri-colour_nxa6050">RunBlue Prestained Molecular-Weight Marker- Tri Color (NXA6050)</a></li> <li><a href="/products/unstained-protein-ladder-14-98-kda-_nbp3-11649">Recombinant Unstained Protein Ladder (NBP3-11649) </a></li> <li><a href="/products/western-blot-protein-standards-12-90-kda-_nbp3-11650">Recombinant Western Blot Protein Standards (NBP3-11650)</a></li> <li><a href="https://www.bio-techne.com/p/staining-kits-reagents/ponceau-s-staining-solution_5225" target="_blank">Ponceau S Staining Solution</a></li> </ul> </td> </tr> </table> <br/><br/> <p align="center"> <a href="/search?keywords=INSTA-Blot"><img src="//images.novusbio.com/design/readyToUse.png" alt="INSTA-blots used for protein screening across multiple tissues for antibody validation." title="Novus provides INSTA-blot membranes for antibody validation" border="0"></a> </p> <br/><br/> <br/><br/> <br/><br/> <br/><br/> </td> </tr> </table> </div></div></div> </div> </div> </div> </div> <div id="block-block-21" class="block block-block"> <div class="content">  <div class="embeddedServiceInvitation" id="snapins_invite" aria-live="assertive" role="dialog" aria-atomic="true" style="display: none;"> <div class="embeddedServiceInvitationHeader" aria-labelledby="snapins_titletext" aria-describedby="snapins_bodytext"> <img id="embeddedServiceAvatar" /> <span class="embeddedServiceTitleText" 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