Real-Time BRET Assays to Measure G Protein/Effector Interactions

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.