Purification of estrogen receptors from MCF-7 human breast cancer cells.
We have partially purified human estrogen receptor from MCF-7 breast cancer cells in order to further characterize the chemical and biological properties of the receptor and to prepare specific receptor antibodies for radioimmunoassay. The estrogen receptor from MCF-7 cell cytosol was purified 1,166-fold (27% recovery) over starting cytosol by combining ammonium sulfate precipitation, affinity chromatography, and Sephadex G-100 gel filtration. The affinity resin consisted of estrone 17-(O-carboxymethyl)oxime:bovine serum albumin: Sepharose 4B. Under high salt conditions, the molecular weight of the purified receptor is 50,000 and is identical to that obtained on chromatography of crude cytosol. Sucrose density gradient centrifugation also revealed the purified receptor sedimenting at the same position as the crude cytosol receptor. The following methods to purify crude cytosol estrogen receptors are much less effective. (a) With DNA-cellulose chromatography, KCl elution revealed peaks at 0.04 M KCl (11-fold purification) and 0.22 M KCl (12.5-fold purification). This could not be combined sequentially with affinity chromatography because high salt conditions were required for affinity elution, and removal of salt by dialysis caused the receptor to adhere to the tubing. (b) With hydroxylapatite chromatography, phosphate elution revealed peaks at 0.12 M phosphate (1.1-fold purification) and 0.175 M phosphate (1.1-fold purification). (c) With phosphocellulose chromatography, KCl elution revealed only one peak at 0.18 M KCl (1.6-fold purification). (d) With diethylaminoethyl cellulose chromatography, KCl elution revealed 4 peaks at 0.025 M (1-fold purification), 0.12 M (1.27-fold purification), 0.14 M (1.28-fold purification), and 0.2 M (0.36-fold purification).
D001943:Breast Neoplasms / Q000032:analysis*; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient / Q000379:methods; D011232:Chemical Precipitation; D002846:Chromatography, Affinity / Q000379:methods; D002848:Chromatography, DEAE-Cellulose / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D006801:Humans; D011960:Receptors, Estrogen / Q000302:isolation & purification*
D011960:Receptors, Estrogen