Determination of BRCA2 oncosuppressor protein expression in human mammary cells by affinity perfusion high-performance chromatography

Abstract

In this work, we report a method for the determination of BRCA2 oncosuppressor protein in human mammary cells by affinity perfusion chromatography. This method involves labeling proteins with [ 35S]-methionine. The isolation and purification of DNA-binding proteins was performed by affinity chromatography on Heparin POROS 20HE. BRCA2 proteins, known to act in the transcriptional control and in DNA repair activity, are included in the DNA-binding proteins. Specific immunoprecipitation was performed with anti-BRCA2 antibodies, and the immune complex [ 35S-BRCA2 proteins/anti-BRCA2 antibodies] was isolated by affinity chromatography on a Protein A POROS column. This procedure allows the determination of the percentage of BRCA2 among the DNA-binding proteins and the quantitation of the difference of expression of BRCA2 oncosuppressor protein in breast carcinoma cells and in normal breast cells treated or untreated with phytoestrogens, such as daidzein or genistein.