Immunoblotting (often referred to as western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. Following electrophoretic separation of proteins and transfer from the gel to an appropriate membrane, the immobilized proteins are probed with specific antibodies to identify and quantitate any antigens present. After being probed with primary antibody, the membrane is washed and the antibody-antigen complexes are identified using horseradish peroxidase (HRPO) or alkaline phosphatase (AP) enzymes coupled to the secondary anti-immunoglobulin-G (anti-IgG) antibody (e.g., goat anti-rabbit IgG). As described in this unit, the detection enzymes are attached directly or via an avidin-biotin bridge to the secondary antibody. Chromogenic or luminescent substrates are also described for visualizing the activity.