CHEMBL3706107

Inhibition Assay: The method employed was adapted from the scientific literature and described in detail by Osboum et al. (1993, Biochemistry, 32, 6229-6236). Recombinant human ERalpha and ERbeta proteins were purified from transfected Sf9-cells. The in vitro assays involved the use of either ERalpha or ERbeta proteins and [3H]E2, at a fixed concentration of 0.5 nM, as the labeled ligand. Recombinant human ERalpha or ERbeta proteins were dissolved in binding buffer (10 mM Tris-HCL, pH 7.5, 10% glycerol, 1 mM DTT, 1 mg/ml BSA) and duplicate aliquots were then incubated with [3H]E2 at a final concentration of 0.5 nM, together with a vehicle control (0.4% DMSO), or the same amount of vehicle containing increasing concentrations of unlabeled steroid ligands as competitors. After incubation for 2 h at 25 C., the unbound ligands were removed and the amounts of [3H]E2 bound to either ERalpha or ERbeta proteins were measured.