Journal
FEBS LETTERS
Volume 531, Issue 2, Pages 245-249Publisher
WILEY
DOI: 10.1016/S0014-5793(02)03508-1
Keywords
fluorescence resonance energy transfer; green fluorescent protein; protein-protein interaction
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Spectral variants of the green fluorescent protein (GFP) have been extensively used as reporters to image molecular interactions in living cells by fluorescence resonance energy transfer (FRET). However, those GFP variants which are the most efficient donor acceptor pairs for FRET measurements show a high degree of spectral overlap which has hampered in the past their use in FRET applications. Here we use spectral imaging and subsequent un-mixing to quantitatively separate highly overlapping donor and acceptor emissions in FRET measurements. We demonstrate the method in fixed and living cells using a novel GFP based FRET pair (GFP2-YFP (yellow)), which has an increased FRET efficiency compared to the most commonly used FRET pair consisting of cyan fluorescent protein and YFP. Moreover, GFP2 has its excitation maximum at 396 nm at which the YFP acceptor is excited only below the detection level and thus this FRET pair is ideal for applications involving sensitized emission. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.
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