Zheng Jie
Department of Physiology and Membrane Biology, University of California at Davis, School of Medicine, USA.
Methods Mol Biol. 2006;337:65-77. doi: 10.1385/1-59745-095-2:65.
The combination of green fluorescent protein mutants and fluorescence resonance energy transfer (FRET) forms a powerful tool for ion channel studies. A key to successful application of green fluorescent protein-based FRET is to reliably separate the FRET signal from various non-FRET fluorescence emissions that coexist in any experimental system. This chapter introduces a FRET quantification method that is based on fluorescence spectroscopic microscopy. Application of this "spectra FRET" method to both the confocal imaging of Xenopus oocytes and the epifluorescence imaging of culture cells is described. The fluorescence intensity ratio measurement, a complementary non-FRET method for identifying the channel subunit stoichiometry, is also discussed.
绿色荧光蛋白突变体与荧光共振能量转移（FRET）相结合，形成了一种用于离子通道研究的强大工具。基于绿色荧光蛋白的FRET成功应用的关键在于，要从任何实验系统中共存的各种非FRET荧光发射中可靠地分离出FRET信号。本章介绍一种基于荧光光谱显微镜的FRET定量方法。文中描述了这种“光谱FRET”方法在非洲爪蟾卵母细胞共聚焦成像和培养细胞落射荧光成像中的应用。还讨论了荧光强度比测量，这是一种用于确定通道亚基化学计量的补充性非FRET方法。
