A comprehensive system for protein purification and biochemical analysis based on antibodies to c-myc peptide

The genomics revolution has created a need for increased speed and generali ty for recombinant protein production systems as well as general methods fo r conducting biochemical assays with the purified protein products. 9E10 is a well-known high-affinity antibody that has found use in a wide variety o f biochemical assays. Here we present a standardized system for purifying p roteins with a simple epitope tag based on c-myc peptide using an antibody affinity column. Antibodies with binding parameters suitable for protein pu rification have been generated and characterized. To purify these antibodie s from serum-containing medium without carrying through contaminating immun oglobulin G, a peptide-based purification process was developed. A fluoresc ence polarization binding assay was developed to characterize the antigen-a ntibody interaction. Protein purification protocols were optimized using a fluorescein-labeled peptide as a surrogate "protein." Binding and elution p arameters were evaluated and optimized and basic operating conditions were defined. Several examples using this procedure for the purification of reco mbinant proteins are presented demonstrating the generality of the system. In all cases tested, highly pure final products are obtained in good yields . The combination of the antibodies described here and 9E10 allow for almos t any biochemical application to be utilized with a single simple peptide t ag. (C) 2001 Academic. Press.