RARalpha Human Retinoic Acid NHR Functional Agonist & Antagonist Coactivator LeadHunter Assay - TW

Procedure Summary:

Human recombinant retinoic acid receptor alpha (RARa) receptors expressed in bacteria are used. Test compound and/or vehicle is preincubated with the 5 nM‡ receptors and binding buffer in modified Tris-HCl buffer pH 7.4 for 16 hours at 4°C. Determination of the amount of complex formed is read spectrofluorimetrically (excitation:337 nm, emission:620/665 nm). Test compound-induced increase in fluorescence by 50 percent or more (≥50%) relative to the 0.1 μM TTNPB response indicates possible RAR a receptor agonist activity. Test compound-induced inhibition of 3 nM TTNPB-induced fluorescence response by 50 percent or more (≥50%) indicates receptor antagonist activity. Note: ‡Membrane protein may change from lot to lot, the concentration used will be adjusted if necessary.

Incubation:

16 hr at 4°C

Control Inhibitor:

Ro41-5253

Control Activator:

TTNPB

Criteria For Significance:

≥ 50% increase in fluorescence relative to TTNPB response, ≥ 50% Inhibition of TTNPB-induced fluorescence

Test Concentration / Dose:

Compounds are screened at 10, 3, 1, 0.3, 0.1, 0.03, 0.01 and 0.003 μM

Test Sample Requirements:

75 μl of 10 mM stock or 1 mg (pre-weighed) powder (assuming FW ≦500 and purity 100%)

Minimum Order Quantity:

16