Characterization of spectral FRET imaging microscopy for monitoring nuclear protein interactions

Authors

  • YE CHEN,

    1. W.M. Keck Center for Cellular Imaging, Departments of Biology and Biomedical Engineering, Gilmer Hall, University of Virginia, Charlottesville, VA 22904, USA
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  • JOSHUA P. MAULDIN,

    1. Departments of Medicine and Cell Biology, University of Virginia Health System, Charlottesville, VA 22908, USA
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  • RICHARD N. DAY,

    1. Departments of Medicine and Cell Biology, University of Virginia Health System, Charlottesville, VA 22908, USA
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  • AMMASI PERIASAMY

    1. W.M. Keck Center for Cellular Imaging, Departments of Biology and Biomedical Engineering, Gilmer Hall, University of Virginia, Charlottesville, VA 22904, USA
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Ammasi Periasamy. Tel: 434 243 7602; fax: 434 982 5210; e-mail: ap3t@virginia.edu

Summary

The spectral processed Förster resonance energy transfer (psFRET) imaging method provides an effective and fast method for measuring protein–protein interactions in living specimens. The commercially available linear unmixing algorithms efficiently remove the contribution of donor spectral bleedthrough to the FRET signal. However, the acceptor contribution to spectral bleedthrough in the FRET image cannot be similarly removed, since the acceptor spectrum is identical to the FRET spectrum. Here, we describe the development of a computer algorithm that measures and removes the contaminating ASBT signal in the sFRET image. The new method is characterized in living cells that expressed FRET standards in which the donor and acceptor fluorescent proteins are tethered by amino acid linkers of specific lengths. The method is then used to detect the homo-dimerization of a transcription factor in the nucleus of living cells, and then to measure the interactions of that protein with a second transcription factor.

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