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Once proteins are separated by gel electrophoresis, most Western blot workflows require transferring the separated proteins from the polyacrylamide gel to a membrane before proteins of interest can be detected. The most effective transfer method is electroblotting, in which an electric field is applied perpendicular to the plane of the gel, causing proteins to migrate to a protein-binding membrane. Electroblotting can be performed using wet and semi-dry transfer systems. Although each system has unique features, they both require building a transfer stack or “sandwich” to ensure that the gel and membrane are pressed closely together. The transfer stack includes the gel, a charged membrane such as nitrocellulose or PVDF and several pieces of filter paper. Western blot transfer also relies on an electrically conductive buffer that often contains methanol or another solvent to promote protein binding and to help remove SDS from the gel. Before building the transfer stac...
