| Assay Method Information | |
| | Muscarinic M2 Receptor Binding Assay |
| Description: | Cell membrane homogenates (60 μg protein) were incubated for 60 minutes at 22° C. with 2 nM [3H]AF-DX 384 in the absence or presence of the test compound in a buffer containing 50 mM Tris-HCl (pH 7.4), 120 mM NaCl, 5 mM KCl, 5 mM MgCl2, and 1 mM EDTA. Nonspecific binding was determined in the presence of 1 μM atropine. Following incubation, the samples were filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters were dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard). The results were expressed as a percent inhibition of the control radioligand specific binding, from which the Ki was calculated. Data were analyzed using software developed at Cerep (Hill software) and validated by comparison with data generated by the commercial software SigmaPlot®4.0 for Windows® (© 1997 by SPSS Inc.). |
| Affinity data for this assay | |
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