Brown Nicole E, Blumer Joe B, Hepler John R
Department of Pharmacology, Emory University School of Medicine, Atlanta, GA, 30322, USA.
Methods Mol Biol. 2015;1278:457-65. doi: 10.1007/978-1-4939-2425-7_30.
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.
生物发光共振能量转移（BRET）是检测蛋白质-蛋白质相互作用的一种有价值的工具。BRET利用具有兼容发射和激发特性的生物发光和荧光蛋白标签，当标签由于蛋白质-蛋白质相互作用而紧密接近（<10 nm）时，能够检测共振能量转移。在这里，我们描述了一种使用海肾荧光素酶和黄色荧光蛋白标签在HEK 293细胞中检测来自两个已知蛋白质结合伴侣（Gαi1和RGS14）的BRET的方案。我们讨论了受体/供体比率以及净BRET的计算，并证明BRET可作为实时研究活细胞中蛋白质-蛋白质相互作用调节的平台。
