[35S]GTPgammaS binding in G protein-coupled receptor assays.
The [(35)S]GTPgammaS 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 alpha(2A)-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 [(35)S]GTPgammaS binding is measured in a 96-well plate format using scintillation counting.
Cooper
Tamara
T
School of Molecular and Biomedical Sciences, University of Adelaide, SA, Australia.
McMurchie
Edward J
EJ
Leifert
Wayne R
WR
D002462:Cell Membrane / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006801:Humans; D011869:Radioligand Assay / Q000379:methods*; D043562:Receptors, G-Protein-Coupled / Q000032:analysis / Q000378:metabolism*; D013462:Sulfur Radioisotopes
D043562:Receptors, G-Protein-Coupled; D013462:Sulfur Radioisotopes; D016244:Guanosine 5'-O-(3-Thiotriphosphate)