FRET is based on the fact that a donor dye (e.g. CFP) in an excited state can transfer a part of its energy through nonradiative dipole–dipole coupling to an acceptor molecule like YFP. The technology involves fusion of donor and acceptor fluorescent proteins to molecules of interest. Co-expression of fusion constructs in living cells enables their interaction to be studied in real time in a quantitative manner. The emission from the acceptor can be detected as soon as both dyes are in proximity, e.g. when interaction of two proteins has taken place.
FRET is used analytically for many assays where the structure of a macromolecule changes or the binding of a ligand to a macromolecule is of interest, especially in the study of.
A limitation of FRET is the requirement for external illumination to initiate the fluorescence transfer which can lead to background noise in the results. This is from direct excitation of the acceptor or due to photobleaching. To avoid this drawback several alternative methods have been developed:
- uses a bioluminescent luciferase as donor resulting in very low background.
- uses a different approach to avoid interference: it uses fluorophores with a very long fluorescence lifetime to avoid the interference caused by molecules with a short fluorescence lifetime or other factors (most importantly, excitation light). The fluorophores of choice are usually chelates of lanthanides (most commonly Europium, Terbium and Samarium). This results in assays of high robustness and sensitivity.
A microplate reader capable of measuring fluorescence with high-sensitivity using filters is the best combination for FRET.can be used, but with a lower performance. It is necessary to perform 2 measurements at different wavelengths, and hence an instrument with at least 2 emission filters is required (this is not possible, for example, in instruments using one single filter cube with 1 excitation filter + 1 emission filter). All microplate readers below are recommended for FRET.