Schematic representation of a hypothetical FRET experiment. In the resting state (left), a transmembrane receptor is fused to cyan fluorescent protein (CFP, donor). Adaptor protein (a) is fused to yellow fluorescent protein (YFP, acceptor). a-YFP is distal from receptor-CFP, so upon excitation at 436 nm, donor fluorescence at 480 nm is recorded. Upon binding to a ligand (L, right), a-YFP binds to receptor-CFP and the reduction in distance enables FRET. Upon equivalent excitation at 436 nm, donor fluorescence (480 nm) is reduced, but acceptor fluorescence at 535 nm is now recorded due to FRET. FRET can similarly be performed with two transmembrane or two soluble factors.