Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser

摘要: One manifestation of fluorescence resonance energy transfer (FRET) is an increase in donor after photobleaching the acceptor. Published acceptor-photobleaching methods for FRET have mainly used wide-field microscopy. A laser scanning confocal microscope enables faster and targeted bleaching within field view, thereby improving speed accuracy. Here we demonstrate approach with CFP YFP, most versatile fluorescent markers now available FRET. CFP/YFP imaging has been accomplished a single (argon) on virtually all laser-scanning microscopes. Accordingly, also describe conditions that developed dual YFP 458 514 argon lines. We detect fusion between signalling molecules (TNF-Receptor-Associated-Factors or TRAFs) are known to homo- heterotrimerize. Importantly, appropriate controls essential avoid false positives by acceptor photobleaching. use two types negative control: (a) internal control (non-bleached areas cell) (b) cells absence (CFP only). find both can yield Given this background, method distinguishing true positive signals. In summary, extensively characterize simple should be adaptable microscopes, its feasibility detecting several partners.