Summary
The [35S]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 Sf9 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 [35S]GTPγS binding is measured in a 96-well plate format using scintillation counting.
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Acknowledgments
The authors thank Mrs. Sharon Burnard, Dr. Olgatina Bucco, and Dr. Amanda Aloia for their assistance.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Cooper, T., McMurchie, E.J., Leifert, W.R. (2009). [35S]GTPγS Binding in G Protein-Coupled Receptor Assays. In: Leifert, W. (eds) G Protein-Coupled Receptors in Drug Discovery. Methods in Molecular Biology, vol 552. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-317-6_10
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DOI: https://doi.org/10.1007/978-1-60327-317-6_10
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