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Related Links Western Blot Protocols, Troubleshooting, & Scientific Resources |
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 cell fractionation), post-translational modifications (e.g. ubiquitination, phosphorylation), protein processing, and protein-protein interactions, when coupled with immunoprecipitation. When sample volume is limited or there is a large workload, the fully automated Simple Western™ platform is a hands-off approach to achieve reproducible results in less time.
Overview of Conventional Western Blot Protocol. (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. Learn more about perfecting Western blots.  Western Blot Primary AntibodiesThe success of a western blot is often dependent upon the specificity of the primary antibody. Novus Biologicals employs the 5 Pillars of Validation 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 by our 100% guarantee and can be tested in unvalidated species with our Risk-Free Testing program. Learn more about primary antibodies. The Simple Western Antibody Database on Bio-Techne.com is a user-interactive listing of all Simple Western validated antibodies to date. Submit your antibody validation data and get a free Separation or Detection Module!
Secondary Antibodies and Detection ReagentsThe 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. Learn more about secondary antibodies.
Western Blot ControlsControls 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 loading controls 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, negative control lysates (knockdown/knockout samples or lysates lacking the expressed protein), or peptide/protein blocking controls to verify that the band(s) representing the protein of interest is(are) present.
Other Western Blotting Support ProductsIn 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.
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