Question

A) David, an undergraduate research assistant in our lab, performed Western Blotting experiment and he used anti- Z protein polyclonal antibody raised in rabbit as a primary antibody. As a secondary antibody, he used anti-mouse HRP labeled antibody. Could you please tell him what is wrong with this experiment?

B) 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

Since we only answer one question at a time as per the honor code, we will answer the first one.

Western blotting is a technique used to separate and identify protein molecules from a mixture of a protein. It is done by first separating the proteins in SDS-PAGE followed by transfer of proteins to a membrane, like nitrocellulose membrane.

Antibodies are the protein molecules that have a role in providing immunity to an organism. Antibodies bind to the specific antigen and direct the immune cells of the body to degrade the antigen. Antibodies bind with antigen through its paratope region located in the Fab region. Fab region consists of both heavy and light chains of the antibody.

When a secondary antibody is used to detect the binding of primary antibody and antigen or just the presence of primary antibody in the system, a secondary antibody conjugated with HRP is used. HRP produces luminescence when reacts with its substrate. Thus, the presence of the primary antibody is detected. Both primary and secondary antibodies have similar heavy chain region as they belong from the same organism or species and thus can easily bind through the heavy chain region.

In this experiment, the primary antibody is from a rabbit and the secondary antibody is from a mouse. Thus, the two antibodies will not be able to bind and no result will be obtained.