1 May 2008 Prolonged irradiation of enhanced cyan fluorescent protein or Cerulean can invalidate Förster resonance energy transfer measurements
Author Affiliations +
J. of Biomedical Optics, 13(3), 031205 (2008). doi:10.1117/1.2937829
Abstract
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 Förster 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.
Birgit Hoffmann, Thomas Zimmer, Nikolaj Klöcker, Laimonas Kelbauskas, Karsten Koenig, Klaus Benndorf, Christoph U. Biskup, "Prolonged irradiation of enhanced cyan fluorescent protein or Cerulean can invalidate Förster resonance energy transfer measurements," Journal of Biomedical Optics 13(3), 031205 (1 May 2008). https://doi.org/10.1117/1.2937829
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KEYWORDS
Luminescence

Proteins

Fluorescence resonance energy transfer

Fluorescent proteins

Capillaries

Streak cameras

Confocal microscopy

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