Dextran-coated charcoal assay
ER preparations were labelled with increasing concentrations of [3H]oestradiol (range 0.25-5 nmol/l) in the absence and presence of a 200-fold excess of unlabelled oestradiol (overnight incubation at 0°C) and unbound ligands were removed using the DCC adsorption method (0.5% charcoal, 0.05% dextran) [29]. Specific binding was calculated from the difference between radioactivity levels measured in the absence and presence of unlabelled oestradiol. Binding capacities were expressed per milligram of protein, the latter being measured using the Bio-Rad assay. Parameters of the binding reaction (n, number of binding sites; Kd, dissociation constant) were estimated using the LIGAND v4.5 program (Dr PJ Munson, National Institutes of Health, Bethesda, MD, USA), and the data were plotted according to the method of Scatchard [30]. The same procedure was applied for [125I]TAZ, except that a 5 nmol/l saturating concentration was used.