title: Reliable measurement of the FRET sensitized-quenching transition factor for FRET quantification in living cells.
abstract: 3-cube-based Förster resonance energy transfer (FRET) microscopy, a sensitized acceptor FRET quantification method, has been widely used to visualize dynamic protein-protein interaction in living cells. Determining the FRET sensitized-quenching transition factor (G factor) of a particular donor-acceptor pair and optical system is crucial for 3-cube FRET quantification. We here improved the acceptor photobleaching-based G factor determination method (termed as mPb-G) and the two-plasmid-based G factor determination method (termed as mTP-G) for rapid and reliable measurement of the G factor. mTP-G method determines G factor by simultaneously detecting three images of cells exclusively expressing each of two tandem constructs with multiple donors and multiple acceptors. This method circumvents switchover of the cells exclusively expressing each of the two constructs. mPb-G method images G factor by detecting three images of cells expressing a donor-acceptor tandem FRET construct before and after partially photobleaching acceptor. We performed the two methods on our dual-channel wide-field FRET microscope to obtain reliable G factor, and also measured the FRET efficiency and acceptor-to-donor concentration ratio of tandem constructs with different acceptor-donor stoichiometries in living HepG2 cells. mTP-G and mPb-G methods provide two simple and reliable tools for determining the G factor, in turn, quantitatively measuring FRET signal and monitoring dynamic biochemical processes in living cells.
authors.affiliation: MOE Key Laboratory of Laser Life Science & College of Life Science, South China Normal University, Guangzhou 510631, China.
authors.forename: Jiang
authors.initials: J
authors.lastname: Zhang
authors.affiliation: MOE Key Laboratory of Laser Life Science & College of Life Science, South China Normal University, Guangzhou 510631, China.
authors.forename: Lili
authors.initials: L
authors.lastname: Zhang
authors.affiliation: MOE Key Laboratory of Laser Life Science & College of Life Science, South China Normal University, Guangzhou 510631, China.
authors.forename: Liuying
authors.initials: L
authors.lastname: Chai
authors.affiliation: MOE Key Laboratory of Laser Life Science & College of Life Science, South China Normal University, Guangzhou 510631, China.
authors.forename: Fangfang
authors.initials: F
authors.lastname: Yang
authors.affiliation: MOE Key Laboratory of Laser Life Science & College of Life Science, South China Normal University, Guangzhou 510631, China.
authors.forename: Mengyan
authors.initials: M
authors.lastname: Du
authors.affiliation: MOE Key Laboratory of Laser Life Science & College of Life Science, South China Normal University, Guangzhou 510631, China. Electronic address: chentsh@scnu.edu.cn.
authors.forename: Tongsheng
authors.initials: T
authors.lastname: Chen
mesh_terms: D036681:Fluorescence Recovery After Photobleaching / Q000295:instrumentation / Q000379:methods; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D056945:Hep G2 Cells / Q000648:ultrastructure; D006801:Humans; D038761:Photobleaching; D010957:Plasmids / Q000235:genetics; D014162:Transfection
keywords: Dual-channel wide-field FRET microscopy; FRET; FRET-sensitized emission; G factor; Living cells