Immunoblotting and immunodetection.

Immunoblotting (often referred to as western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit presents procedures for electrophoretically transferring antigens from a denaturing polyacrylamide gel in a tank or a semidry transfer apparatus to a nitrocellulose, PVDF, or nylon membrane. The process can be monitored by reversible staining or by Ponceau S staining, both of which are described here. A protocol for blotting previously stained gels is also described. The transferred proteins are bound to the surface of the membrane, providing access for reaction with immunodetection reagents. All remaining binding sites are blocked by immersing the membrane in a solution containing either a protein or detergent blocking agent. After probing with the primary antibody, the membrane is washed and the antibody-antigen complexes are identified with horseradish peroxidase (HRPO) or alkaline phosphatase enzymes coupled to the secondary anti-IgG antibody (e.g., goat anti-rabbit IgG). The enzymes are attached directly or via an avidin-biotin bridge to the secondary antibody, and protocols are provided for both methods. Chromogenic or luminescent substrates are then used as described to visualize the activity. Finally, a method for stripping and reprobing membranes is presented.