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Sci. STKE, 18 April 2006
Vol. 2006, Issue 331, p. re2
Steven S. Vogel*,
Christopher Thaler, and
Srinagesh V. Koushik
National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Laboratory of Molecular Physiology, 5625 Fishers Lane, Bethesda, MD 20892, USA.
Gloss: Förster resonance energy transfer (FRET) is used to measure protein-protein interactions in living cells. When FRET occurs, a "donor" fluorophore is excited by the absorption of a photon, and that energy is transferred by a nonradiative mechanism to a nearby acceptor molecule. The fraction of energy-transfer events relative to donor excitation events is called the FRET efficiency. It has a steep dependence on the separation distance between the donor and acceptor. Proteins of interest tagged with spectral variants of green fluorescent protein (GFP) can be readily used for FRET studies in living cells. The proper interpretation of FRET measurements, however, requires an understanding of the limitations of the numerous methods used for measuring FRET, as well as consideration of the subtleties of FRET theory, particularly its dependence on the abundance of donors and acceptors. This review highlights aspects of the acquisition of FRET measurements, and of FRET theory, that are vital for proper interpretation. The adoption of "standards" with known FRET efficiencies is a first step toward eliminating erroneous interpretations of FRET experiments due to errors in the accuracy and precision of FRET measurements.