alpha2A Human Adrenoceptor GPCR GTPgammaS Agonist & Antagonist LeadHunter Assay - TW

Procedure Summary:

Human recombinant adrenergic α2A receptors expressed in Chem-1 cells are used. Test compound is preincubated with 0.018 mg/ml‡ membranes and 3 µM GDP for 20 minutes at 25°C in modified HEPES buffer pH 7.4, then SPA beads are added for another 60 minutes at 30°C. The reaction is initiated by 0.3 nM [35S]GTPγS for an additional 30 minute incubation period. Test compound-induced increase of [35S]GTPγS binding by 50 percent or more (≥50%) relative to 10 µM UK 14,304 response indicates possible adrenergic α2A receptor agonist activity. Test compound-induced inhibition of 0.1 µM UK 14,304-induced increase of [35S]GTPγS binding response by 50 percent or more (≥50%) indicates receptor antagonist activity. Compounds are screened at 10, 1, 0.1, 0.01 and 0.001 µM. Note: ‡Membrane protein may change from lot to lot, the concentration used will be adjusted if necessary.

Incubation:

30 min at 30°C

Control Inhibitor:

Yohimbine

Control Activator:

Brimonidine

Criteria For Significance:

≥ 50% Increase in bound [35S]GTPgammaS relative to UK-14304 response, ≥ 50% Inhibition of UK-14304-induced bound [35S]GTPgammaS

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