Abstract
Bioluminescence resonance energy transfer (BRET) is a valuable tool to detect protein-protein interactions. BRET utilizes bioluminescent and fluorescent protein tags with compatible emission and excitation properties, making it possible to examine resonance energy transfer when the tags are in close proximity (<10 nm) as a typical result of protein-protein interactions. Here we describe a protocol for detecting BRET from two known protein binding partners (Gαi1 and RGS14) in HEK 293 cells using Renilla luciferase and yellow fluorescent protein tags. We discuss the calculation of the acceptor/donor ratio as well as net BRET and demonstrate that BRET can be used as a platform to investigate the regulation of protein-protein interactions in live cells in real time.