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Proceedings Paper

FRET microscopy to visualize transcription factor dimerization in the nucleus of the living cell
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Paper Abstract

Cells respond to environmental cues or developmental programs by modifying protein complexes in the nucleus to alter patterns of gene transcription. Recent advances in digital imaging coupled with the development of new fluorescent probes provide the tools to begin to study where and when changes in protein interactions take place in the nucleus of the living cell. Here, we describe the application of fluorescence resonance energy transfer (FRET) using both wide-field and 2-photon (2P) microscopy to visualize the interactions of the transcription factor CAATT/enhancer binding protein alpha (C/EBP(alpha) ) in living pituitary cells. The efficiency of FRET will be improved if the overlap of the donor emission spectra with the absorption spectra for the acceptor is increased. The trade off for this improved efficiency, however, is that there will be an increase in the background signal from which the weak sensitized acceptor emission must be extracted. Here, we compare and contrast the FRET signals obtained from dimerized C/EBP(alpha) proteins fused to several different color variants of the jellyfish green fluorescent protein (GFP). We use both wide-field and 2P FRET microscopy to characterize the spectral cross-talk and FRET signals for each of the donor and acceptor pairs.

Paper Details

Date Published: 24 April 2001
PDF: 8 pages
Proc. SPIE 4262, Multiphoton Microscopy in the Biomedical Sciences, (24 April 2001); doi: 10.1117/12.424550
Show Author Affiliations
Richard N. Day, Univ. of Virginia (United States)
Ammasi Periasamy, Univ. of Virginia (United States)


Published in SPIE Proceedings Vol. 4262:
Multiphoton Microscopy in the Biomedical Sciences
Ammasi Periasamy; Peter T. C. So, Editor(s)

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