期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 104, 期 24, 页码 10264-10269出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0701987104
关键词
C/EBP; COP1; FRET; luminescence; fluorescence
资金
- NIGMS NIH HHS [R01 GM 065467, R01 GM065467] Funding Source: Medline
FRET is a well established method for cellular and subcellular imaging of protein interactions. However, FRET obligatorily necessitates fluorescence excitation with its concomitant problems of photobleaching, autofluorescence, phototoxicity, and undesirable stimulation of photobiological processes. A sister technique, bioluminescence resonance energy transfer (BRET), avoids these problems because it uses enzyme-catalyzed luminescence; however, BRET signals usually have been too dim to image effectively in the past. Using a new generation electron bombardment-charge-coupled device camera coupled to an image splitter, we demonstrate that BRET can be used to image protein interactions in plant and animal cells and in tissues; even subcellular imaging is possible. We have applied this technology to image two different protein interactions: (i) dimerization of the developmental regulator, COP1, in plant seedlings; and (ii) CCAAT/ enhancer binding protein alpha (C/EBP alpha) in the mammalian nucleus. This advance heralds a host of applications for imaging without fluorescent excitation and its consequent limitations.
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