title: Measuring FRET using time-resolved FLIM.
abstract: Cell migration is a process that is controlled by the formation and correct localization of protein complexes and by post-translational modification of individual proteins. Forster or fluorescent resonance energy transfer (FRET) detected using fluorescence lifetime imaging microscopy (FLIM) provides a method by which protein-protein interactions may be detected and spatially localized within a cell. This technique can be used to map protein activation states and the formation and dissolution of protein complexes that control movement of a cell. This chapter describes a protocol for detecting FRET between GFP- and mRFP1-tagged proteins in fixed adherent cells. A background to both FRET and FLIM is provided followed by an overview of the method and a full protocol for sample preparation, data acquisition, and analysis.
authors: Randall Division of Cell and Molecular Biophysics, King’s College London, London, UK.
authors: Penny E
authors: PE
authors: Morton
authors: Maddy
authors: M
authors: Parsons
mesh_terms: D045744:Cell Line, Tumor; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012984:Software; D014162:Transfection
chemical_list: D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins