kappa (KOP) Human Opioid GPCR GTPgammaS Agonist & Antagonist LeadHunter Assay - TW

Procedure Summary:

Human recombinant opiate κ receptor stably expressed in Chem-1 cells are used. Test compound and/or vehicle is preincubated with the membranes and GDP in modified HEPES pH 7.4 buffer for 20 minutes at RT and SPA beads are then added for another 60 minutes at 30°C. The reaction is initiated by [³⁵S]GTPγS for an additional 30 minutes incubation period. Test compound-induced increase of [³⁵S]GTPγS binding by 50 percent or more (≥50%) relative to the 10 µM U-69593 response indicates possible opiate κ receptor agonist activity. Test compound-induced inhibition of 0.1 µM U-69593-induced increase of [³⁵S]GTPγS binding response by 50 percent or more (≥50%) indicates possible receptor antagonist activity.

Incubation:

30 min at 30°C

Control Inhibitor:

nor-Binaltorphimine

Control Activator:

U-69593

Criteria For Significance:

≥ 50% Increase in bound [35S]GTPgammaS relative to U-69593 response, ≥ 50% Inhibition of U-69593-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