A western blot and immunoprecipitation assay to verify antibody specificity

Abstract

Life scientists are raising awareness of the problem of irreproducible data in basic research (1) (2). Part of the crisis of irreproducibility as been blamed on antibodies (3), and from this standpoint, an equal push to bring awareness to antibody validation has been initiated (4) (5) (6) (7). Although scientists agree the onus of validation is on the user, it is also viewed that commercial antibody providers should share in this responsibility (4) (6) (8) (7). For companies who try to provide breadth in their catalog, validation is not inconsequential. It presents a difficult and costly impediment; nevertheless, users deserve transparency and a genuine effort from vendors to qualify their products. The first and most important quality of validation is verifying the antibody recognizes the intended target. The most accepted methods for validating antibody specificity include application-specific testing using knockout (KO) or RNA knockdown samples and/or using multiple antibodies against distinct epitopes of the intended target (6) (9). This paper describes Bethyl Laboratories’ solution for a practical, high-throughput method for validating the target-specificity of antibodies for the application of western blot (WB). The method involves verification of specificity via WB of immunoprecipitates using two or more antibodies that recognize distinct epitopes of a target protein.