In FRET assays, interaction between biomolecules is measured indirectly by conjugating one of a pair of carefully selected fluorescent dyes to each of the molecules of interest. When these fluorescent dyes are held in close proximity due to binding of the biomolecules, a unique fluorescence signal is developed that specifically confirms the proximity and thus the binding reaction. The degree of proximity and the geometry of the molecules determine the extent to which energy transfer takes place. When energy transfer is complete, fluorescence of the donor molecule is completely extinguished, and all fluorescence appears at the emission wavelength of the acceptor. In proximity assays, less complete quenching of donor fluorescence can be expected, but it is the appearance of acceptor fluorescence in response to excitation of the donor molecule that indicates that the molecular interaction of interest has taken place.
Most synthetic fluors consist of a single fluorophore.
The acceptor molecule should be minimally excited by the wavelength employed to excite the donor. Controls demonstrate that the fluorescence response observed was due solely to proximity based on the streptavidin-biotin binding reaction. RPE-biotin fluorescence at 660 nm for 495 nm excitation was subtracted as a blank from each value. FRET finds utility in any application where the proximity of two conjugatable molecules is of interest.