M3 Human Acetylcholine (Muscarinic) GPCR Binding Antagonist Radioligand LeadHunter Assay - TW

Procedure Summary:

Human recombinant muscarinic M3 receptors expressed in CHO-K1 cells are used in modified Tris-HCl buffer pH 7.4. A 12 µg‡ aliquot is incubated with 0.8 nM [³H]N-Methylscopolamine for 120 minutes at 25℃. Non-specific binding is estimated in the presence of 1 µM atropine. Receptors are filtered and washed, the filters are then counted to determine [³H]N-Methylscopolamine specifically bound. Compounds are screened at 10 µM.
Note: ‡Membrane protein may change from lot to lot, the concentration used will be adjusted if necessary.

Ligand:

[³H] N-Methylscopolamine

Ligand Kd (nM):

0.75

Ligand Concentration:

0.8 nM

Non Specific:

1 µM Atropine

Incubation:

120 min at 25°C

Control Inhibitor:

4-Damp methiodide

Criteria For Significance:

≥ 50% of max stimulation or inhibition

Test Concentration / Dose:

Suggested concentration for initial testing is 10 uM

Test Sample Requirements:

Minimum for 1) Screen: 25 μl of 10 mM stock -OR- 1 mg (pre-weighed). 2) Dose Response: 60 μl of 10 mM stock -OR- 1 mg (pre-weighed).

Minimum Order Quantity:

2