Principle and quantitative assessment of FRET via DFRAP. (a) In case of FRET between the donor CFP and the acceptor YFP due to interaction between two proteins A and B, the photochemical destruction of the acceptor abolishes FRET and leads to an increased emission from the donor, CFP. CFP and YFP are depicted as cyan and yellow ribbon models fused to putative interacting proteins A and B respectively. (b, c). Time-course analysis of fluorescence intensity before and after photobleaching in the presence or absence of a protein-protein interaction. Blue and yellow curves indicate the levels of CFP and YFP fluorescence before and after photobleaching, respectively. In case of FRET, bleaching of the acceptor molecule leads to an increase in donor fluorescence (b). In the absence of interaction between proteins A and B, CFP levels before and after the bleach do not vary considerably (c). BB – Before bleach, AB – After bleach.