Prolonged irradiation of enhanced cyan fluorescent protein or Cerulean can invalidate Forster resonance energy transfer measurements

被引:22
|
作者
Hoffmann, Birgit [1 ]
Zimmer, Thomas [1 ]
Kloecker, Nikolai [3 ]
Kelbauskas, Laimonas [1 ]
Koenig, Karsten [2 ]
Benndorf, Klaus [1 ]
Biskup, Christoph [1 ]
机构
[1] Univ Klinikum Jena, Inst Physiol 2, D-07740 Jena, Germany
[2] Univ Saarland, Fac Phys & Mechtron, D-66123 Saarbrucken, Germany
[3] Univ Freiburg, Inst Physiol, D-79104 Freiburg, Germany
关键词
enhanced cyan fluorescent protein; Cerulean; time-resolved spectroscopy; streak camera; Forster resonance energy transfer; fluorescence resonance energy transfer; fluorescence lifetime imaging; photobleaching; photoconversion;
D O I
10.1117/1.2937829
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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. (c) 2008 Society of Photo-Optical Instrumentation Engineers.
引用
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页数:9
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