M4 Human Acetylcholine (Muscarinic) GPCR GTPgammaS Agonist & Antagonist LeadHunter Assay - TW

Procedure Summary:

Human recombinant Muscarinic M4 receptors expressed in CHO-K1 cells are used. Test compound is preincubated with membranes and GDP for 20 minutes at RT in modified HEPES buffer pH 7.4 with SPA beads are added for another 60 minutes at 30°C. The reaction is then initiated by addition of [³⁵S]GTPγS for 15 minutes. Test compound-induced increase of [³⁵S]GTPγS binding by 50 percent or more (≥50%) relative to the 300 µM McN-A-343 response indicates possible Muscarinic M4 agonist activity. Test compound-induced inhibition of 10 µM McN-A-343-induced increase of [³⁵S]GTPγS binding response by 50 percent or more (≥50%) indicates receptor antagonist activity.

Incubation:

15 min at 30°C

Control Inhibitor:

4-Damp methiodide

Control Activator:

McN-A-343

Criteria For Significance:

≥ 50% Increase in bound [35S]GTPgammaS relative to McN-A-343 response, ≥ 50% Inhibition of McN-A-343-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