| Solution Information | help | |
| Enzyme: | Muscarinic acetylcholine receptor M3 | |
| inhibitor: | BDBM43821 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | The purpose of this screen was to test compounds of interest against the muscarinic M3 receptor for their ability to modulate [3H]N-methyl-scopolamine ([3H]-NMS) binding. Experimental Protocol: Membranes were prepared from M3-expressing CHO cells as described (Shirey et al., 2008). Binding reactions contained 0.1 nM [3H]-NMS, 20 ug of membranes and compound or vehicle (0.3% DMSO, final, to define total binding) or 1uM atropine (to define nonspecific binding) in a total volume of 500 ul. The KD of [3H]-NMS was determined empirically to be 0.21 nM. Compounds were serially diluted in DMSO, then diluted in assay buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) to give a final DMSO concentration of 0.3% in the binding reaction. Binding reactions were incubated for 2 hours at room temperature on a Lab-Line Titer plate shaker at setting 7 (~750 rpm). Reactions were stopped and membranes collected onto 96-well Barex microplates with GF/B filter (1um pore size) using a Brandel harvester | |
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