| Assay Method Information | |
| | Estrogen Receptor Binding Assay |
| Description: | Small molecules were tested for estrogen receptor (ER) alpha binding using a modification of the LanthaScreen TR-FRET Estrogen Receptor Alpha Coactivator Assay (Thermofisher cat # A15885) where the fluorescein-labeled coactivator peptide was replaced with Fluormone ES2 Green (Thermofisher cat # PV6045) to improve assay signal. Briefly, compounds were serially diluted in DMSO then transferred into assay buffer (Thermofisher cat # PV4295+5 mM DTT) at a 1:10 dilution. 10 μl of compound was transferred to a 96 half-area black well plate (Corning cat #3694) in duplicate. 5 μl of ER-LBD protein (5 nM stock in assay buffer; Thermofisher cat #4542) was added to each well. In addition 5 μl of a prepared stock of Fluormone ES2 Green (12 nM) and terbium-labeled anti-GST monoclonal antibody (mAb) (8 nM; Thermofisher cat # PV3550) in assay buffer was also added to each well. Plates were incubated at room temperature (RT) for 4 hours, and then TR-FRET emission ratio was measured using an EnVision Multilabel Plate Reader (Perkinelmer #2104-0010). |
| Affinity data for this assay | |
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