Detecting protein-protein interactions with CFP-YFP FRET by acceptor photobleaching.
FRET is a light microscopy method for detecting protein-protein interactions within intact cells. The FRET protocol presented here is for CFP- and YFP-tagged proteins examined with an argon laser on a scanning confocal microscope. FRET is assayed by one of the most straightforward approaches available, namely, acceptor photobleaching. In this procedure, the YFP-tagged protein (the FRET "acceptor") is photobleached at a cellular site of interest, and then the intensity of the CFP-tagged protein (the FRET "donor") at that same site is measured. In principle, FRET is detected when the CFP intensity increases after the photobleaching of YFP. This unit describes the appropriate steps to perform this measurement, as well as the necessary controls to ensure that an increase in CFP intensity, if detected, in fact reflects bona fide FRET. Successful application of the protocol will support the conclusion that the CFP- and YFP-tagged proteins directly interact at the site of the photobleaching.
Karpova
Tatiana
T
National Institutes of Health, Bethesda, Maryland, USA.
McNally
James G
JG
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002468:Cell Physiological Phenomena; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; D011506:Proteins; D011994:Recombinant Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins