Prolonged irradiation of enhanced cyan fluorescent protein or Cerulean can invalidate Forster resonance energy transfer measurements.
Since its discovery, green fluorescent protein (GFP) and its variants have proven to be a good and convenient fluorescent label for proteins: GFP and other visible fluorescent proteins (VFPs) can be fused selectively to the protein of interest by simple cloning techniques and develop fluorescence without additional cofactors. Among the steadily growing collection of VFPs, several pairs can be chosen that can serve as donor and acceptor fluorophores in Forster resonance energy transfer (FRET) experiments. Among them, the cyan fluorescent proteins (ECFP/Cerulean) and the enhanced yellow fluorescent protein (EYFP) are most commonly used. We show that ECFP and Cerulean have some disadvantages despite their common use: Upon irradiation with light intensities that are commonly used for intensity- and lifetime-based FRET measurements, both the fluorescence intensity and the fluorescence lifetime of ECFP and Cerulean decrease. This can hamper both intensity- and lifetime-based FRET measurements and emphasizes the need for control measurements to exclude these artifacts.
Hoffmann
Birgit
B
Universitatsklinikum Jena, Institut fur Physiologie II, Kollegiengasse 9 07740 Jena, Germany.
Zimmer
Thomas
T
Klöcker
Nikolaj
N
Kelbauskas
Laimonas
L
König
Karsten
K
Benndorf
Klaus
K
Biskup
Christoph
C
D016477:Artifacts; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000378:metabolism* / Q000528:radiation effects; D008027:Light; D008657:Metabolic Clearance Rate / Q000528:radiation effects; D008856:Microscopy, Fluorescence / Q000379:methods*; D011829:Radiation Dosage; D012680:Sensitivity and Specificity
C485182:Cyan Fluorescent Protein; D049452:Green Fluorescent Proteins