Protein Blotting: Immunoblotting

Immunoblotting (also referred to as Western blotting) uses antibodies to probe for a specific protein in a sample bound to a membrane. Typically, a protein sample is first size separated via electrophoresis (e.g., SDS PAGE). However, antibodies used for specific protein detection are restricted by the polyacrylamide gel and, to make the separated proteins accessible, the proteins need to be moved out of the gel and bound to a rectangular sheet of PVDF or nitrocellulose membrane. In this second step, the membrane, cut to the same dimensions of the SDS gel (e.g., 10 x 10 cm), is then laid on the gel surface. The gel and membrane sandwich is then positioned in specialized blotting equipment that electrophoretically transfers the negatively charged proteins from the gel onto the membrane. The nitrocellulose or PVDF membrane binds the proteins as they move out of the gel, producing an exact replica, on the membrane surface, of the original protein gel separation. The proteins bind with high capacity and, in contrast to the polyacrylamide gel, are freely accessible to antibody reagents. The membrane is then blocked to prevent any nonspecific protein binding and visualized by specific antibodies to detect the presence or absence of a particular protein. For routine quantitation of a protein, the SDS PAGE separation is not always needed, and whole cell lysates or other complex mixtures are bound directly to the membrane for analysis using slot or dot blotting. Applications of immunoblotting are many, and include antibody characterization, diagnostics, gene expression and post translational modification analysis.