High Background from Insufficient Antibody Specificity

Symptom

High background signal observed in immunoprecipitation experiments where multiple non-target protein bands appear in the eluate, indicating poor antibody specificity.

1. 1 Antibody preparation contains non-specific antibodies or polyspecific antibodies
2. 2 Antibody has not been affinity-purified against the target antigen
3. 3 Antibody contains cross-reactive antibodies recognizing unrelated epitopes
4. 4 Crude serum or ascites used instead of purified antibody preparation

1. 1 Use affinity-purified antibody that has been purified against the target antigen
2. 2 Select pre-adsorbed antibody preparations to remove cross-reactive species
3. 3 Validate antibody specificity using knockout or knockdown cell lysates as negative controls
4. 4 Switch to monoclonal antibodies if using polyclonal preparations with high background