title: Bioluminescence resonance energy transfer to detect protein-protein interactions in live cells.
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.
authors: Department of Pharmacology, Emory University School of Medicine, Atlanta, GA, 30322, USA.
authors: Nicole E
authors: NE
authors: Brown
authors: Joe B
authors: JB
authors: Blumer
authors: John R
authors: JR
authors: Hepler
mesh_terms: D060807:Bioluminescence Resonance Energy Transfer Techniques; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D049410:Luciferases, Renilla / Q000737:chemistry; D008163:Luminescent Measurements; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D020710:RGS Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D020710:RGS Proteins; C491400:RGS14 protein, human; D049410:Luciferases, Renilla; D019206:GTP-Binding Protein alpha Subunits, Gi-Go