Isolation, purification and quantification of BRCA1 protein from tumour cells by affinity perfusion chromatography

A new procedure for the isolation, purification and quantification of the product of the oncosuppressor gene brca1 in breast tissues, was carried out. It involves internal cell protein [35S]methionine labelling followed by two perfusion chromatographies. The first one is heparin affinity chromatography, to purify all of the cell DNA-binding proteins. A subsequent specific immunoprecipitation of BRCA1 protein was performed with an antibody raised against BRCA1. The immune complex was isolated using the second chromatographic step, Protein A affinity chromatography. The amount of BRCA1 expressed by cells was expressed as a ratio, in percent, calculated as follows: 100x amount of labelled DNA-binding proteins (dpm) that bound specifically to the anti-BRCA1 polyclonal antibodies (K-18)/amount of whole labelled DNA-binding protein (dpm) purified on a heparin column. Applications to MCF7 and T-47D human breast tumour cell lines, which were treated or not using 2 mM sodium butyrate demonstrated an increase in BRCA1 protein expression.