| Assay Method Information | |
| | In vitro RORc Ligand Binding Assay |
| Description: | On day of the assay, 100 μL of 0.05% CHAPS (in deionized H2O) was added to all wells of the GFB Unifilter plate and allowed soak for 1 h. A wash buffer of 50 mM HEPES (pH 7.4), 150 mM NaCl, and 5 mM MgCl2 was prepared to wash the filter plate. To prepare an assay buffer, BSA was added to the wash buffer to reach 0.01% and DTT was added to reach 1 mM. For IC50 mode, 10 mM compound stocks were serially diluted in DMSO with DMSO to give 20× required final concentration in DMSO (15 μL compound + 30 μL DMSO). The 20× compound stocks were diluted in DMSO with Assay Buffer 4-fold to reach 5× the final test concentration in 25% DMSO (10 μL compound + 30 μL Assay Buffer). Solutions were mixed by aspiration several times with a pipette set on 50 μL volume. For the assay, 10 μL of 5× compound stock solutions in 25% DMSO were added to the assay plate in duplicate. Assay plates were 96-well polypropylene V-bottom plates. 10 μL of 5× compound in 25% DMSO/75% Assay Buffer was added to Test wells. 10 μL of 25% DMSO/75% Assay Buffer was added to Total Binding or No Receptor wells. 10 μL of 5 μM 25-hydroxycholesterol in 25% DMSO/75% Assay Buffer was added to NSB wells. 20 μL of 15 nM 25-[3H]hydroxycholesterol prepared in Assay Buffer was added to all wells. 20 μL of 1.5 ug/mL RORc receptor was added to wells (or 40 μL Assay Buffer to No R wells). Following addition to the wells, the plates were incubated 3 h at 25 °C. Final concentrations were as follows: 50 mM HEPES buffer (pH 7.4); 150 mM NaCl; 1 mM DTT; 5 mM MgCl2; 0.01% BSA; 5% DMSO; 0.6 ug/mL RORc receptor; 6 nM 25-[3H]hydroxycholesterol. For NSB wells, 1 μM 25-hydroxycholesterol was also present. |
| Affinity data for this assay | |
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