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.
Hizel
C
C
Laboratoire d'Oncologie Moléculaire, INSERM CRI 9502 & EA 2145, Centre Jean Perrin, Clermont-Ferrand, France.
Maurizis
J C
JC
Rio
P
P
Communal
Y
Y
Chassagne
J
J
Favy
D
D
Bignon
Y J
YJ
Bernard-Gallon
D J
DJ
D019313:BRCA1 Protein / Q000032:analysis / Q000302:isolation & purification*; D001943:Breast Neoplasms / Q000737:chemistry*; D002087:Butyrates / Q000494:pharmacology; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000032:analysis / Q000302:isolation & purification; D005434:Flow Cytometry; D006801:Humans; D011233:Precipitin Tests; D014407:Tumor Cells, Cultured
D019313:BRCA1 Protein; D002087:Butyrates; D004268:DNA-Binding Proteins