| Solution Information | help | |
| Enzyme: | Estrogen receptor beta | |
| inhibitor: | BDBM48441 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Compounds identified as active from the primary screen, AID 633 - HTS of Estrogen Receptor-beta Coactivator Binding Inhibitors, were selected for testing in this assay. Each compound was tested in 6 concentration, 1:2 serial dilutions starting at a nominal test concentration of 50 micromolar. Each concentration was tested in triplicate. Reagents: 1. Assay buffer: 20 mM Tris buffer, pH 7.5, 50 mM NaCl, and 0.01% NP40 2. ER-beta protein: Biotin-labeled ER-beta-LBD protein 3. 17 beta-estradiol 4. Lance Eu-W1024 labeled-streptavidin (SA-Eu) 5. SRC-Cy5 peptide 6. Positive Control: SRC1-BOX II peptide Procedure: 1. Make assay reaction buffer for HTS that contains recombinant ER-beta protein (0.3 nM, final concentration), SRC-Cy5 peptide (2 nM, final concentration), 17 beta-estradiol (1 uM, final concentration) and SA-Eu (0.5 nM, final concentration). 2. Dispense 19 uL of assay reaction buffer to 384-well black assay plate. 3. Add library compound (1 mM in DMSO) to each well and i | |
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