Quantitative fluorescence resonance energy transfer (FRET) measurement with acceptor photobleaching and spectral unmixing.
Fluorescence resonance energy transfer (FRET) by acceptor photobleaching is a simple but effective tool for measurements of protein-protein interactions. Until recently, it has been restricted to qualitative or relative assessments owing to the spectral bleed-through contamination resulting from fluorescence overlap between the donor and the acceptor. In this paper, we report a quantitative algorithm that combines the spectral unmixing technique with FRET by acceptor photobleaching. By spectrally unmixing the emissions before and after photobleaching, it is possible to resolve the spectral bleed-through and retrieve the FRET efficiency/interaction distance quantitatively. Using a human keratinocyte cell line transfected with cyan fluorescent protein (CFP)- and yellow fluorescent protein (YFP)-tagged Cx26 connexins as an example, FRET information at homotypic gap junctions is measured and compared with well-established methods. Results indicate that the new approach is sensitive, flexible, instrument independent and solely FRET dependent. It can achieve FRET estimations similar to that from a sensitized emission FRET method. This approach has a great advantage in providing the relative concentrations of the donor and the acceptor; this is, for example, very important in the comparative study of cell populations with variable expression levels.
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Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, London WC2A 3PX, U.K. yan.gu@csc.mrc.ac.uk
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Kelsell
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D000465:Algorithms; D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis; D002460:Cell Line; D000072259:Connexin 26; D017630:Connexins; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis; D018613:Microscopy, Confocal / Q000379:methods; D011994:Recombinant Proteins / Q000032:analysis; D014162:Transfection
D001426:Bacterial Proteins; D017630:Connexins; C000607042:GJB2 protein, human; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C054562:yellow fluorescent protein, Bacteria; D000072259:Connexin 26; D049452:Green Fluorescent Proteins