Dot blot assay for determining fusion protein antiserum quality

When testing antiserum quality, it is essential to determine first whether the antiserum recognizes the fusion protein against which it was raised. A dot blot will provide a qualitative estimate of antibody titer, as described in this protocol. The same test can be done quantitatively by enzyme-linked immunosorbent assay (ELISA), but the technically simpler dot blot gives adequate information, uses less protein, and does not require an ELISA plate reader. If the same protein has been expressed in two different fusion-protein vectors, the test should be performed with the protein not used for immunization. If there is only one fusion protein-e.g., a glutathione-S-transferase (GST)-fusion-a second dot blot can be done in parallel with GST alone to compare the titers. If the antiserum recognizes the protein, there should be a lower titer of antibodies against GST alone as compared to the entire fusion protein. A preimmune control is always advisable. If a positive result is not obtained against the fusion protein, it is futile to proceed to secondary tests. Continue boosting and retesting until a positive response is achieved, or try a different approach.