Progestin-receptor analysis in human breast cancer cytosol by isoelectric focusing in slabs of polyacrylamide gel

Abstract

A method is described for the analysis of progestin receptor in human breast cancer cytosol by isoelectric focusing in slabs of polyacrylamide gel. The synthetic progestin [ 3H]-R 5020 was used as a ligand. The progestin receptor focused at pH 6.2 and was separated from non-specifically bound [ 3H]-R 5020 that focused at pH 5.0–5.4 (contaminating protein from serum). A comparison to sucrose gradient centrifugation showed a good correlation between the two methods. Both the 8 S complex and the 4 S complex seen in tumor cytosols focused at pH 6.2. Limited trypsin digestion resulted in disappearance of the 8 S complex and increase in the 4 S complex seen by sucrose gradient centrifugation. Further proteolysis resulted in the formation of an R 5020-binding complex sedimenting in the 4 S region which was not possible to analyse by isoelectric focusing. The time needed for focusing of the progestin receptor was 1.5h. Conditions were established where only one incubation was needed per receptor analysis reducing the amount of tissue required.