iii. Blocking and Addition of Antibodies

At this point, the proteins in the SDS PAGE gel have been transferred laterally to the nitrocellulose membrane from the gel. Now, primary and secondary antibodies are used to probe the nitrocellulose membrane for the specific protein you are looking for. This is done through a series of incubations, where the membrane is soaked in solutions containing the appropriate antibodies.

The steps in this procedure are:

• Blocking the membrane, by incubating the membrane in blocking solution (see below). This step puts protein on the membrane where none was transferred:
• Incubate the membrane in the primary antibody. The primary antibody sticks to the protein of interest.
• Wash the membrane three times (for five minutes each) with the wash solution (a modified version of the blocking solution), to eliminate any unbound primary antibody.
• Incubate the membrane in the specially modified secondary antibody . The secondary antibody binds to the primary antibody molecules.
• Rinse the membrane with modified blocking solution three times for five minutes each.
  

The blocking solution is a 2% solution of BSA (bovine serum albumin) in PBS (phosphate buffered saline). The modified blocking solution has a small amount of surfactant ( a detergent) added.

What’s going on?

The blocking step keeps the antibodies from sticking to exposed areas of the membrane by coating these areas with protein (from the BSA). The primary antibody binds to the protein of interest. The secondary antibody binds to the primary antibody, and has been specially modified to allow visualization.

To wash the membrane after addition of primary and secondary antibodies, the blocking solution is used, adding 5 milliliters of it to the culture dish containing the membrane. The culture dish is rocked on the rocker table for 5 minutes and then the blocking solution is removed. This process is repeated a total of three times.

The secondary antibody is added after the wash step. The secondary antibody has two important properties: it binds specifically to the primary antibody, and it contains an enzyme that is involved in the development stage of the western blotting technique.