Prolonged irradiation of enhanced cyan fluorescent protein or Cerulean can invalidate Förster resonance energy transfer measurements
- 1 January 2008
- journal article
- Published by SPIE-Intl Soc Optical Eng in Journal of Biomedical Optics
- Vol. 13 (3), 031205-031205-9
- https://doi.org/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.Keywords
This publication has 25 references indexed in Scilit:
- Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactionsJournal of Microscopy, 2007
- A guide to choosing fluorescent proteinsNature Methods, 2005
- An improved cyan fluorescent protein variant useful for FRETNature Biotechnology, 2004
- Interaction of PSD-95 with potassium channels visualized by fluorescence lifetime-based resonance energy transfer imagingJournal of Biomedical Optics, 2004
- Expansion of the Genetic Code Enables Design of a Novel “Gold” Class of Green Fluorescent ProteinsJournal of Molecular Biology, 2003
- High-Order Photobleaching of Green Fluorescent Protein inside Live Cells in Two-Photon Excitation MicroscopyBiochemical and Biophysical Research Communications, 2002
- Fluorescence resonance energy transfer microscopy: a mini reviewJournal of Biomedical Optics, 2001
- THE GREEN FLUORESCENT PROTEINAnnual Review of Biochemistry, 1998
- Aequorea green fluorescent proteinFEBS Letters, 1994
- Primary structure of the Aequorea victoria green-fluorescent proteinGene, 1992