Spectral imaging and linear un-mixing enables improved FRET efficiency with a novel GFP2-YFP FRET pair.
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.
Zimmermann
Timo
T
Advanced Light Microscopy Facility, European Molecular Biology Laboratory, Meyerhofstr. 1, Heidelberg, Germany.
Rietdorf
Jens
J
Girod
Andreas
A
Georget
Virginie
V
Pepperkok
Rainer
R
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000737:chemistry
D001426:Bacterial Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins