期刊
BIOPHYSICAL CHEMISTRY
卷 127, 期 3, 页码 155-164出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bpc.2007.01.008
关键词
CFP; YFP; fluorescence; lifetime; proteins
资金
- Engineering and Physical Sciences Research Council [GR/S96555/01] Funding Source: researchfish
We have used widefield photon-counting FLIM to study FRET in fixed and living cells using control FRET pairs. We have studied fixed mammalian cells expressing either cyan fluorescent protein (CFP) or a fusion of CFP and yellow fluorescent protein (YFP), and living fungal cells expressing either Cerulean or a Cerulean-Venus fusion protein. We have found the fluorescence behaviour to be essentially identical in the mammalian and fungal cells. Importantly, the high-precision FLIM data is able to reproducibly resolve multiple fluorescence decays, thereby revealing new information about the fraction of the protein population that undergoes FRET and reducing error in the measurement of donoracceptor distances. Our results for this simple control system indicate that the in vivo FLIM-FRET studies of more complex protein-protein interactions would benefit greatly from such quantitative measurements. (c) 2007 Elsevier B.V. All rights reserved.
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