Fluorescent proteins have become a mainstay in the study of protein localization, protein-protein interaction and gene expression. By rendering a protein of interest natively fluorescent, fluorescent proteins allow the intracellular localization and quantitation of that protein without invasive labeling procedures. Multicolor applications for fluorescent proteins include fluorescent protein-paired fluorescence resonance energy transfer.
Fluorescence resonance energy transfer, or FRET, provides the means for examining the proximity of molecules within the range of 10 to 100 angstroms. In FRET studies (Figure 1), a donor fluorophore is optimally excited and, if a suitably chosen acceptor fluorophore is within the specified distance, the donor fluorescence emission excites the acceptor fluorophore and the acceptor fluorophore fluorescence is seen. If FRET occurs, donor fluorescence should decrease in intensity, while acceptor fluorescence should increase. When FRET is used in conjunction with the MoFlo high performance cell sorter, researchers can acquire information about molecular proximity at rates exceeding 100,000 data points/second. They also can collect cells of interest at rates approaching 70,000 cells/second.