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Measurement of FRET Efficiency and Ratio of Donor to Acceptor Concentration in Living Cells

Huanmian Chen ^*, Henry L. Puhl, 3rd ^*, Srinagesh V. Koushik , Steven S. Vogel  and Stephen R. Ikeda ^*

Laboratory of Molecular Physiology, Sections on ^* Transmitter Signaling and Cellular Biophotonics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland 20892

Correspondence: Address reprint requests and inquiries to Stephen R. Ikeda, Tel.: 301-443-2807; Fax: 301-480-0466; E-mail: sikeda{at}mail.nih.gov.

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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