| Assay Method Information | |
| | Competitive Radioligand-Binding Assay |
| Description: | Estrogen receptor (ERβ) binding affinity of the NRBAs was also determined using an in vitro competitive radioligand-binding assay with [3H]-estradiol ([3H]-E2, PerkinElmer), a high affinity ligand for both ERα and ERβ. The equilibrium dissociation constant (Kd) of [3H]-E2 was determined by incubating increasing concentrations of [3H]-E2 (0.01 to 10 nM) with bacterially expressed ERα or ERβ ligand binding domain (LBD) at 4° C. for 18 hours (h). Non-specific binding was determined by adding 1000 nM E2 to the incubation mixture. It was determined that the minimum concentration of [3H]-E2 required to saturate ERα and ERβ binding sites in the incubation mixture was 1 nM, respectively. The binding affinity of the NRBAs was determined under identical conditions by incubating increasing concentrations (3×10−2 to 1,000 nM) of ligand with isolated ER LBD and 1 nM [3H]-E2. Following incubation, bound and free [3H]-E2 were separated by using vacuum filtration with the Harvester (PerkinElmer). Briefly, the incubation mixture was filtered through a high affinity protein binding filter, and washed several times to remove any unbound radioactivity. The filter plate was air dried and sealed on the bottom. Scintillation cocktail was added to each well and the top of the plate was sealed. Radioactivity was counted in a TopCount NXT Microplate Scintillation Counter. |
| Affinity data for this assay | |
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