Question

Immunoblotting can be used to detect a protein of interest.

Two proteins of interest are found in the same cells. Propose an
experiment that uses immunoblotting to compare the relative quantity of
these two proteins.

Answer 1

Immunoblotting is based on specific antigen-antibody interactions. The primary antibody we use should be specific to our protein of interest, while the secondary antibody should be specific for the primary antibody. If we have two proteins of interest in the same cells (sample),

1. Run SDS-PAGE with samples,

2. Transfer the proteins from the gel to membrane (blot),

3. Do immunoblotting with primary antibody specific for the first protein and detect the results,

4. Strip the primary antibody for the first protein with stripping buffer,

5. Do immunoblotting with primary antibody specific for the second protein and detect the results in the same blot.

Using stripping buffer, we can reuse the same blot for 2-4 times for 2-4 proteins of interest.