Unit

UNIT 12.10 Detecting Protein-Protein Interactions In Vivo with FRET using Multiphoton Fluorescence Lifetime Imaging Microscopy (FLIM)

  1. David Llères,
  2. Samuel Swift,
  3. Angus I. Lamond

Published Online: 1 OCT 2007

DOI: 10.1002/0471142956.cy1210s42

Current Protocols in Cytometry

Current Protocols in Cytometry

How to Cite

Llères, D., Swift, S. and Lamond, A. I. 2007. Detecting Protein-Protein Interactions In Vivo with FRET using Multiphoton Fluorescence Lifetime Imaging Microscopy (FLIM). Current Protocols in Cytometry. 42:12.10:12.10.1–12.10.19.

Author Information

  1. Wellcome Trust Biocentre, College of Life Sciences, University of Dundee, United Kingdom

Publication History

  1. Published Online: 1 OCT 2007
  2. Published Print: OCT 2007

Abstract

Protein interactions are critical for many processes in mammalian cells. Such interactions include the stable association of proteins within multi-subunit complexes and the transient association of regulatory proteins. Information about protein interactions in cells has previously come from either in vitro analyses using recombinant expressed proteins, or from yeast 2–hybrid studies. A limitation of this approach is that the protein interaction is studied in isolation, without regard to the many competing protein interactions that can occur within cells. This unit presents a light microscopy approach for detecting protein-protein interactions in vivo based on the measurement of FRET using the multiphoton fluorescence lifetime imaging microscopy (FLIM) technique. By using the FLIM-FRET technique, the spatial organization and quantification of such interactions in a living cell can be characterized. A detailed protocol describing the complete microscope procedure and the choice of the appropriate experimental controls as well as the FRET calculations is also included. Curr. Protocol. Cytom. 42:12.10.1-12.10.19. © 2007 by John Wiley & Sons, Inc.

Keywords:

  • fluorescence lifetime;
  • GFP;
  • mCherry;
  • FRET;
  • FLIM;
  • multiphoton;
  • protein interaction