Estrogen synthetase (aromatase). Affinity purification of antibody against the cytochrome P450 component

Abstract

To procure an affinity gel capable of purifying antibody against the cytochrome P450 component of estrogen synthetase (P450_ES), we coupled purified P450_ES to agarose supports. Our purpose was to compare two differently-activated agarose gels (Affi-Gel 15 and Tresyl-activated Sepharose) with the same P450_ES preparation to compare the efficiency of coupling and the yield of purified antibody. Using supplier-directed protocols to covalently attach P450_ES to each of the supports, and identical procedures to bind and elute anti-P450_ES, we reached the following conclusions. Tresyl-activated Sepharose bound greater amounts of antigen than Affi-Gel 15 based on the amount of residual antigen after the coupling procedure and the amount of bound antigen detected by an ELISA-type method. However, both ligand-coupled supports yielded comparable amounts of purified anti-P450_ES at a 48-fold purification relative to the starting IgG preparation.