Western Blot Protocol
Western blotting (WB) is the most widely performed immunoassay and is the best initial validation technique used to identify proteins of interest within a tissue homogenate or cell extract. In WB, proteins are first separated based on size (molecular weight) via gel electrophoresis. The resulting gel is then placed next to a membrane, made of nitrocellulose or polyvinylidene fluoride (PVDF), and the application of an electrical current induces the proteins to transfer from the gel to the membrane. The membrane can then be probed with antibodies that are specific for a target protein of interest. Lastly, the membrane is exposed to secondary antibodies and detection reagents enabling visualization.