Reliable measurement of the FRET sensitized-quenching transition factor for FRET quantification in living cells |
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| Institution: | 1. College of Mechanical and Electrical Engineering, Central South University, Changsha, China;2. Department of Systems Engineering and Engineering Management City University of Hong Kong, Hong Kong, China;3. School of Mechanical Science & Engineering, Huazhong University of Science and Technology, China;4. Guangdong Engineering Research Center for Green Manufacturing & Energy Efficiency Optimization, Guangdong University of Technology, Guangzhou 510006, China;1. State Key Laboratory of Solidification Processing, School of Materials Science and Engineering, Northwestern Polytechnical University, Xi''an 710072, PR China;2. Key Laboratory of Radiation Detection Materials and Devices of Ministry of Industry and Information Technology, Northwestern Polytechnical University, Xi''an 710072, PR China;3. Multidisciplinary Material Research Center, Frontier Institute of Science and Technology, Xi’an Jiaotong University, Xi''an 710049, PR China;1. Physics and Astronomy, University of Nebraska, Kearney, NE, 68849, USA;2. Biomedical Engineering Institute, Old Dominion University, Norfolk, VA, 23529, USA;3. Institute of Micro and Nano technology, Old Dominion University, Norfolk, VA, 23529, USA;4. Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA, 23529, USA;1. Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, VA, USA;2. Department of Forensic Science, Virginia Commonwealth University, Richmond, VA, USA;1. Cancer and Inflammation Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA;2. Sackler Inst. of Molecular Medicine, Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel |
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| 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. |
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| Keywords: | FRET Dual-channel wide-field FRET microscopy FRET-sensitized emission Living cells |
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