Quantitative determination of nuclear estrogen receptors by an enzyme immunoassay: applicability and caveats.
A method is presented with which approx. 95% of nuclear estrogen receptors appear to be extracted from MCF-7 cells. Since both nuclear isolation and nuclear estrogen receptor extraction take place in a single test tube with only vortex mixing, loss of nuclear material is minimized. The amount of nuclear estrogen receptors in the nuclear extract was determined by direct [3H]estradiol labeling of monolayer cultures and with a commercially available estrogen receptor immunoassay (ER-EIA) kit. Since the ER-EIA kit was designed and calibrated for quantitative determination of cytosolic estrogen receptor isolated in low ionic strength buffer, the applicability of the ER-EIA to quantitative determination of estrogen receptor content in high ionic strength nuclear extraction buffer was tested. A linear relationship exists between the amount of nuclear estrogen receptor detected by the immunoassay, the amount of receptor present in serial dilutions of the nuclear extract and the amount of nuclear estrogen receptor detected in cells by [3H]estradiol labeling of monolayer cultures, the absolute amount of nuclear estrogen receptors determined by the immunoassay consistently exceeded the amount of receptor detected by [3H]estradiol labeling. The possibility that the enzyme immunoassay must be properly calibrated for the specific conditions of the nuclear estrogen receptor assay is discussed.
D015153:Blotting, Western; D001943:Breast Neoplasms / Q000032:analysis*; D002021:Buffers; D002467:Cell Nucleus / Q000032:analysis*; D003600:Cytosol / Q000032:analysis; D004958:Estradiol / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D017830:Octoxynol; D009994:Osmolar Concentration; D011092:Polyethylene Glycols; D011189:Potassium Chloride; D011960:Receptors, Estrogen / Q000032:analysis*; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D002021:Buffers; D011960:Receptors, Estrogen; D012710:Serum Albumin, Bovine; D011092:Polyethylene Glycols; D004958:Estradiol; D011189:Potassium Chloride; D017830:Octoxynol; C010615:Nonidet P-40