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Flow Cytometry Protocols pp 371–392Cite as

Flow Cytometric FRET Analysis of Protein Interaction

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 699))

Abstract

Investigation of protein–protein interactions in situ in living or intact cells gains expanding importance as structure/function relationships proposed from bulk biochemistry and molecular modeling experiments require demonstration at the cellular level. Fluorescence resonance energy transfer (FRET)-based methods are excellent tools for determining proximity and supramolecular organization of biomolecules at the cell surface or inside the cell. This could well be the basis for the increasing popularity of FRET; in fact, the number of publications exploiting FRET has doubled in the past 5 years. In this chapter, we intend to provide a generally useable protocol for measuring FRET in flow cytometry. After a concise theoretical introduction, recipes are provided for successful labeling techniques and measurement approaches. The simple, quenching-based population-level measurement; the classic ratiometric, intensity-based technique providing cell-by-cell actual FRET efficiencies, and a more advanced version of the latter, allowing for cell-by-cell autofluorescence correction, are described. Finally, points of caution are given to help design proper experiments and critically interpret the results.

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Acknowledgments

The authors were supported by the following grants: EU FP6 LSHBCT-2004-503467, LSHC-CT-2005-018914, MRTN-CT-2005-019481, and MCRTB-CT-035946; Hungarian National Research Fund K62648, K75752, K68763, K72677; Hungarian National Development Agency TAMOP-4.2.2-08/1-2008-0019 and TAMOP-4.2.11B-09/11KONV-2010-0007; and Hungarian Ministry of Health ETT 362/2009.

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Authors and Affiliations

  1. Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary

    György Vereb, Péter Nagy & János Szöllo˝si

Authors
  1. György Vereb
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  2. Péter Nagy
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  3. János Szöllo˝si
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Corresponding author

Correspondence to György Vereb .

Editor information

Editors and Affiliations

  1. Medical Center, Flow Cytometry Core Facility, George Washington University, I Street NW 2300, Washington, 20037, District of Columbia, USA

    Teresa S. Hawley

  2. Medical Center, Dept. Anatomy & Regenerative Biology, George Washington University, I Street NW 2300, Washington, 20037, District of Columbia, USA

    Robert G. Hawley

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Vereb, G., Nagy, P., Szöllo˝si, J. (2011). Flow Cytometric FRET Analysis of Protein Interaction. In: Hawley, T., Hawley, R. (eds) Flow Cytometry Protocols. Methods in Molecular Biology, vol 699. Humana Press. https://doi.org/10.1007/978-1-61737-950-5_18

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  • DOI: https://doi.org/10.1007/978-1-61737-950-5_18

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  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61737-949-9

  • Online ISBN: 978-1-61737-950-5

  • eBook Packages: Springer Protocols

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