| Assay Method Information | |
| | Binding Activity of Compound of the Present Invention to Estrogen Receptor ER |
| Description: | The binding activity of the compound to ER was determined using the LanthaScreen TR-FRET ER Alpha Coactivator Assay kit (brand: Thermo, Cat. No: A15885). The ER receptor agonist Estradiol (brand: Sigma, Cat. No: E8875-25) was diluted 10-fold with DMSO in a 96-well V-bottom plate. The highest concentration was 100 μM, 8 concentrations in total. The test compound was diluted 10-fold with DMSO. The highest concentration was 3000 μM, 8 concentrations in total. The compound was then further diluted 50-fold with the detection buffer Nuclear Receptor Buffer E (containing 5 mM DTT) provided in the kit, 10 μL of the diluted compound was transferred to a 96-well half-area microplate (brand: Corning, Cat. No: 3694), 5 μL of ER-LBD protein (4× concentration) was added to the test compound, then a mixture of 5 μL of fluorescein-coactivator peptide and Tb-labeled anti-GST antibody (4× concentration) was added, and the experiment was performed in duplicate. The plate was incubated at room temperature for 2 h, the fluorescence values (Excitation 337, Emission 520/495 nm) were detected using the PHERAstar instrument, and the 520:495 ratio was calculated. With the log value of the final concentration of the compound as the X axis and the 520/495 ratio as the Y axis, data was input into the software Graphpad Prism 9, and the EC50 value was calculated by four-parameter fitting. |
| Affinity data for this assay | |
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