title: Real-time BRET assays to measure G protein/effector interactions.
abstract: Advances in imaging assays based on resonance energy transfer (RET) have made it possible to study protein/protein interactions in living cells under physiological conditions. It is now possible to measure the kinetics of changes in these interactions in response to ligand stimulation in real time. Here we describe protocols for these assays focusing on the basal and ligand-stimulated interaction between tagged Gβγ subunits and adenylyl cyclase II. We describe relevant positive and negative controls and various experimental considerations for optimization of these experiments.
authors.affiliation: Department of Pharmacology and Therapeutics, McGill University, Montréal, QC, Canada.
authors.forename: Darlaine
authors.initials: D
authors.lastname: Pétrin
authors.forename: Mélanie
authors.initials: M
authors.lastname: Robitaille
authors.forename: Terence E
authors.initials: TE
authors.lastname: Hébert
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D060807:Bioluminescence Resonance Energy Transfer Techniques / Q000379:methods*; D002460:Cell Line; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D025941:Protein Interaction Mapping / Q000379:methods*
chemical_list: D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases