Laboratory of Biomolecular Network Dynamics, Biochemistry, Molecular and Structural Biology Section, Department of Chemistry, KU Leuven, Heverlee, Belgium.
Methods Mol Biol. 2022;2525:207-218. doi: 10.1007/978-1-0716-2473-9_15.
Bioluminescence resonance energy transfer (BRET) has gained impetus to monitor protein interactions in proximity. BRET involves the energy transfer from a bioluminescent donor (luciferases) to a fluorescent acceptor. Since bioluminescence is an intrinsic phenomenon, BRET excludes the need for external illumination and serves as a powerful alternative to fluorescence-based systems. However, BRET has not been widely adopted for single-cell imaging applications, mainly due to the low signal output resulting in poor signal-to-noise ratio. In this chapter, we describe a protocol to optimize spatiotemporal BRET imaging by adopting fluorescent HaloTag acceptors, adapting cell culture conditions and microscopic setup.
生物发光共振能量转移（BRET）在监测近距离蛋白质相互作用方面得到了推动。BRET 涉及从生物发光供体（荧光素酶）到荧光受体的能量转移。由于生物发光是一种内在现象，BRET 不需要外部照明，是荧光基系统的有力替代品。然而，BRET 尚未广泛应用于单细胞成像应用，主要是由于信号输出低导致信噪比差。在本章中，我们描述了通过采用荧光 HaloTag 受体、适应细胞培养条件和显微镜设置来优化时空 BRET 成像的方案。
