Detecting protein-protein interactions in vivo with FRET using multiphoton fluorescence lifetime imaging microscopy (FLIM)

David Llères, Samuel Swift, Angus I. Lamond

    Research output: Other contribution

    35 Citations (Scopus)

    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.

    Original languageEnglish
    TypeLab Protocol
    PublisherJohn Wiley & Sons Inc.
    ISBN (Electronic)9780471142959
    DOIs
    Publication statusPublished - Oct 2007

    Keywords

    • Fluorescence Resonance Energy Transfer
    • Image Processing, Computer-Assisted
    • Microscopy
    • Multiphoton
    • Fluorescence
    • Protein Interaction Mapping
    • Protein Subunits
    • Proteins

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