Removal of nonspecific binding proteins is required in co-immunoprecipitation with nuclear proteins

METHOD SUMMARYCo-immunoprecipitation is a common method for investigation of protein-protein interactions. However, according to published protocols, it is unclear whether the protein samples should be pretreated with the beads to remove nonspecific binding proteins. The authors investigated the effects of bead types, ion concentrations, NP-40 and pretreatment steps on nonspecifically bound proteins to minimize false positives. Whether protein samples should be pretreated to remove nonspecific binding proteins in co-immunoprecipitation (CO-IP) is controversial. In this work, nonspecific binding of proteins to agarose beads was found to be greater than that to magnetic beads. The nonspecific binding was increased with the decrease of ion concentrations but reduced by Nonidet P40. Western blot indicated that p65 and beta-actin were present as nonspecifically bound protein to the beads. p53 and beta-actin were present in the CO-IP precipitates of nuclear proteins but pretreatment cleared the nonspecifically pulled down p53 and beta-actin. These data suggest that magnetic beads are better for CO-IP, but preclearing is necessary to minimize false positive regardless of which bead is used, particularly for nuclear proteins.