| Solution Information | help | |
| Enzyme: | Estrogen receptor | |
| inhibitor: | BDBM42013 | |
| 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 629 - HTS of Estrogen Receptor-alpha Coactivator Binding Inhibitors, were selected for testing in this assay. Reagents 1.Assay buffer: 50 mM TRIS pH 8.0, 10% Glycerol, 50 mM KCL, 0.1% Bovine Serum Albumin, 0.05% NP40. Wash Buffer: 1X PBS , 0.05% Tween 20. 2.Biotin SRC-3 (supplied by PI), Estrogen Receptor alpha mouse mAb-Cell Signalling cat #62A3, Estrogen Receptor beta mAb- ABR cat # MA1-23221, Protein A/G coated microplate-Pierce cat# 1858788. Streptavidin-HRP-Jackson Immunoresearch- cat# 016-030-084. SuperBlock -Pierce cat # 37535. 3.Human Recombinant Estrogen Receptor alpha- Invitrogen cat # PV-P2187, Human Recombinant Estrogen Receptor beta cat # PV-2466, Estradiol-Sigma cat #E-1024, 0.1N Sulfuric acid cat # 319589. 4.ALR-136D as positive control: 1 mM stock in DMSO. Procedure 1.Preincubate estrogen receptor α or β diluted 1:150 with 4uM estradiol in Assay Buffer for 30 min on ice. 2.Add 25uL/well of m | |
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