High Background from Insufficient Antibody Specificity (Immunoprecipitation (High Background))

Question

Accepted Answer

High background signal observed in immunoprecipitation experiments where multiple non-target protein bands appear in the eluate, indicating poor antibody specificity. Common causes: 1) Antibody preparation contains non-specific antibodies or polyspecific antibodies 2) Antibody has not been affinity-purified against the target antigen 3) Antibody contains cross-reactive antibodies recognizing unrelated epitopes 4) Crude serum or ascites used instead of purified antibody preparation Solutions: 1) Use affinity-purified antibody that has been purified against the target antigen 2) Select pre-adsorbed antibody preparations to remove cross-reactive species 3) Validate antibody specificity using knockout or knockdown cell lysates as negative controls 4) Switch to monoclonal antibodies if using polyclonal preparations with high background