Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy

Fluorescence resonance energy transfer (FRFT) detects the proximity of fluo rescently labeled molecules over distances > 100 Angstrom. When performed i n a fluorescence microscope, FRET can be used to map protein-protein intera ctions in vivo. We here describe a FRET microscopy method that can be used to determine whether proteins that are colocalized at the level of light mi croscopy interact with one another. This method can be implemented using di gital microscopy systems such as a confocal microscope or a wide-field fluo rescence microscope coupled to a charge-coupled device (CCD) camera. It is readily applied to samples prepared with standard immunofluorescence techni ques using antibodies labeled with fluorescent dyes that act as a donor and acceptor pair for FRET. Energy transfer efficiencies are quantified based on the release of quenching of donor fluorescence due to FRET, measured by comparing the intensity of donor fluorescence before and after complete pho tobleaching of the acceptor. As described, this method uses Cy3 and Cy5 as the donor and acceptor fluorophores, but can be adapted for other FRET pair s Including cyan fluorescent protein and yellow fluorescent protein, (C) 20 01 Academic Press.