title: Detecting protein-protein interactions in vivo with FRET using multiphoton fluorescence lifetime imaging microscopy (FLIM).
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.
authors.affiliation: Wellcome Trust Biocentre, College of Life Sciences, University of Dundee, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Llères
authors.forename: Samuel
authors.initials: S
authors.lastname: Swift
authors.forename: Angus I
authors.initials: AI
authors.lastname: Lamond
mesh_terms: D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007091:Image Processing, Computer-Assisted / Q000379:methods; D008853:Microscopy / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods; D025941:Protein Interaction Mapping / Q000379:methods; D021122:Protein Subunits / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*
chemical_list: D021122:Protein Subunits; D011506:Proteins