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Measurement of FRET Efficiency and Ratio of Donor to Acceptor Concentration in Living Cells
Open accessAbstract
Measurement of fluorescence resonance energy transfer (FRET) efficiency and the relative concentration of donor and acceptor fluorophores in living cells using the three-filter cube approach requires the determination of two constants: 1), the ratio of sensitized acceptor emission to donor fluorescence quenching (G factor) and 2), the ratio of donor/acceptor fluorescence intensity for equimolar concentrations in the absence of FRET (k factor). We have developed a method to determine G and k that utilizes two donor-acceptor fusion proteins with differing FRET efficiencies-the value of which need not be known. We validated the method by measuring the FRET efficiency and concentration ratio of the fluorescent proteins Cerulean and Venus in mammalian cells expressing a series of fusion proteins with varying stoichiometries. The method greatly simplifies quantitative FRET measurement in living cells as it does not require cell fixation, acceptor photobleaching, protein purification, or specialized equipment for determining fluorescence spectra or lifetime.
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Cited by
Fluorescent protein FRET: the good, the bad and the ugly
Piston, D. W., & Kremers, G.-J. (2007) · Trends in Biochemical Sciences · doi:10.1016/j.tibs.2007.08.003 · Cited by 849
Accuracy and precision in quantitative fluorescence microscopy
Waters, J. C. (2009) · Journal of Cell Biology · doi:10.1083/jcb.200903097 · Cited by 613
Förster resonance energy transfer – A spectroscopic nanoruler: Principle and applications
Sahoo, H. (2011) · Journal of Photochemistry and Photobiology C: Photochemistry Reviews · doi:10.1016/j.jphotochemrev.2011.05.001 · Cited by 405
Abdollahi, A., Roghani-Mamaqani, H., & Razavi, B. (2019) · Progress in Polymer Science · doi:10.1016/j.progpolymsci.2019.101149 · Cited by 300
Single Cell Optical Imaging and Spectroscopy
Stender, A. S., Marchuk, K., Liu, C., Sander, S., Meyer, M. W., Smith, E. A., Neupane, B., Wang, G., Li, J., Cheng, J.-X., et al. (2013) · Chemical Reviews · doi:10.1021/cr300336e · Cited by 292
Metadata
- Authors
- Chen, H., Puhl, H. L., Koushik, S. V., Vogel, S. S., & Ikeda, S. R.
- Venue
- Biophysical Journal
- Publisher
- Elsevier BV
- Published
- 2006-09
- Volume
- 91
- Issue
- 5
- Pages
- L39-L41
- ISSN
- 0006-3495 (ISSN-L)
- OpenAlex
- W1988949448
- Cited by
- 263
- References
- 0
- FWCI
- 6.78
- Type
- journal article
- Language
- en
- Open access
- bronze
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- Chen, H., Puhl, H. L., Koushik, S. V., Vogel, S. S., & Ikeda, S. R. (2006). Measurement of FRET Efficiency and Ratio of Donor to Acceptor Concentration in Living Cells. Biophysical Journal. https://doi.org/10.1529/biophysj.106.088773
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