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        [35S]GTPS Binding in G Protein-Coupled Receptor Assays

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        The [35 S]GTPγS binding assay to measure G protein activation following agonist binding to G protein-coupled receptors (GPCRs) remains a powerful molecular technique to substantiate traditional pharmacological values of potency, efficacy, and affinity. The method described uses membrane preparations of the α2A -adrenergic receptor and purified G protein subunits expressed in Sf 9 cells, reconstituted into a functional signaling system. This technology is generic and could be used with other GPCRs to demonstrate initial signaling events following receptor activation. Agonist-stimulated [35 S]GTPγS binding is measured in a 96-well plate format using scintillation counting.
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