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| | Radioligand Binding Assay |
| Description: | In the GR radioligand binding assay, test compounds were serially diluted in semi-log steps (10 concentrations) with a final concentration of 10 μM. Test compounds (1 μL) and controls (1 μL) in 100% DMSO were added to 96 Greiner V-bottom polypropylene plates. 0% control was 6.7% DMSO (final concentration in assay) and 100% control was 6.7 μM Dexamethasone.The full length GR was diluted to a final concentration of 3.3% (0.495 mg/ml) in assay buffer (20 mM Tris-HCl, 1 mM EDTA, 10% (w/v) Glycerol, 20 mM Sodium molybdate, pH 7.4). 45 μL of GR was added to each well and the plates were incubated for 15 min at room temperature. 3H-dexamethasone solution was diluted to a concentration of 70 nM in assay buffer (7 nM final assay concentration) and 5 μL was added to each well. The samples were mixed for 5 min using a plate shaker at 700 rpm, before incubation for 2 h at room temperature.50 μL ice-cold charcoal solution (pH 7.4: 2% Charcoal, 0.2% Dextran T70 in 20 mM Tris-HCl, 1 mM EDTA and 20 mM Sodium molybdate) was added to each well and the samples were mixed on plate shaker for 5 minutes. |
| Affinity data for this assay | |
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