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Assay Method Information

Assay Name:  [3H]NMS/Carbachol Binding Assay
Description:  In brief, binding reactions containing ~10 μg of membrane protein per tube were carried out for 2 h (22 C) in 1 ml of binding buffer containing 25 mM sodium phosphate and 5 mM MgCl2 (pH 7.4). In saturation binding assays, we employed six different [3H]NMS concentrations (200 to 7,000 pM). In competition binding assays, we used a fixed concentration of [3H]NMS (500 pM) in the presence of 10 different concentrations of carbachol, the cold competitor. Nonspecific binding was defined as binding observed in the presence of 10 μM atropine. Binding reactions were terminated by rapid filtration over GF/C Brandel filters, followed by three washes (~4 ml/wash) with ice-cold distilled water. The amount of radioactivity that remained bound to the filters was determined by liquid scintillation spectrometry.
Affinity data for this assay
 

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Last update November 1, 2007
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