Question

David, an undergraduate intern in our lab, forget to block the blot with BSA and continue to the antibody hybridization steps and finally developed his blot. Could you please describe his blot picture without even looking!

Answer 1

The blot will have so many non-specific bands and high background signal as a result it will be not possible to detect the band of the target protein.

Explanation:

Blocking is a very important step in western blotting experiment. During the western blotting, the proteins are transferred into a membrane. The membrane has an ability to bind with the proteins. Because of the fact that the antibodies are also proteins, they also has the tendency to bind with the membrane. So after transferring the target protein into the membrane, it is necessary to block the other sites on the membrane to avoid non-specific binding of the antibody. Bovine serum albumin (BSA) is usually used as the blocking solution and it will bind with all the places in the membrane other than where the target protein is bound. Hence, the antibody added will not be able to bind with the membrane but bind specifically to the target protein. If the blocking is not done, then the antibody will bind non-specifically to the membrane creating lot of background signals in the final blot.