Last updated: 1/12/12 By: Tim Starr
Follow this protocol to detect proteins on a PVDF or nitrocellulose filter. The first step is to "block" the membrane, which means to soak the membrane in a buffer containing a lot of miscellaneous protein (like albumin in milk or Bovine serum albumin). The purpose is to cover any part of the membrane that will bind non-specifically to proteins, that way when you add the antibody specific to your protein it will only bind to your protein and not to other parts of the membrane by accident. The second step is to soak the membrane in an antibody that is specific for the protein you want to detect. The third step is to soak the antibody in a secondary antibody that binds to the primary antibody. This secondary antibody will be attached to an enzyme such as HRP or a fluorescent molecule, which can be used for detection. The last step is to soak the membrane in the substrate solution and then image the membrane
Wash Buffer: Tris-Buffered Saline with Tween20 (TBS-T)