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Assay Method Information

Assay Name:  [35S]GTPγS Binding/Immunoprecipitation Assay
Description:  In brief, membranes from COS-7 cells expressing different Cys-substituted mutant M3R constructs were first treated with CuPhen (100 μM; 10 min at 37 C). Control samples were incubated under the same experimental conditions in the absence of CuPhen. Subsequently, receptor-containing membranes (75 μg of protein) were incubated with the muscarinic agonist, carbachol (1 mM) for 2 min at 30 C in the presence of 1 nM [35S]GTPγS. Following membrane solubilization and pre-clearing, Gαq/11 proteins were immunoprecipitated with a selective anti-Gαq/11-specific antiserum as described [Akam et al., Br. J. Pharmacol., 132:950-958]. Nonspecific binding was determined in the presence of 10 μM GTPγS.
Affinity data for this assay
 

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Last update November 1, 2007
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