H1 Human Histamine GPCR GTPgammaS Agonist & Antagonist LeadHunter Assay - TW

Procedure Summary:

Human recombinant histamine H1 receptors expressed in CHO-K1 cells are used. Test compound is preincubated with 0.032 mg/ml‡ receptor and 1 μM GDP in modified HEPES buffer pH 7.4 for 20 minutes at 25°C, SPA beads are then added for another 60 minutes at 30°C. The reaction is initiated by 0.3 nM [35S]GTPgS for an additional 30 minute incubation period. Test compound-induced increase of [35S]GTPγS binding by 50 percent or more (≥50%) relative to 1 mM histamine response indicates possible histamine H1 agonist activity. Test compound-induced inhibition of 3 μM histamine-induced increase of [35S]GTPgS binding response by 50 percent or more (≥50%) indicates receptor antagonist activity.
‡Membrane protein may change from lot to lot, the concentration used will be adjusted if necessary.

Incubation:

30 min at 30°C

Control Inhibitor:

Pyrilamine

Control Activator:

Histamine

Criteria For Significance:

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