Imaging protein-protein interactions by Fluorescence Resonance Energy Transfer (FRET) microscopy.
Detection of specific protein-protein interactions has long been restricted to bulk biochemical methods such as immunoprecipitation and immunoblotting. Even more sensitive methods using general immunofluorescence are limited, and it is difficult to infer protein-protein interactions from the results of these tests. Fluorescence Resonance Energy Transfer (FRET) is a photophysical process that can be exploited to obtain highly sensitive information about such interactions. It can sense the presence of acceptor fluorophores in the vicinity of a donor fluorophore within a separation distance that is the size of a single protein molecule. This unit details FRET microscopy based on release of quenched donor fluorescence after acceptor photobleaching, microinjection of reagents into the nucleus or cytosol, and labeling of antibodies for these procedures.
Wouters
Fred S
FS
Imperial Cancer Research Fund, London, United Kingdom.
Bastiaens
Philippe I H
PI
D000818:Animals; D002232:Carbocyanines / Q000378:metabolism; D002478:Cells, Cultured; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D014162:Transfection / Q000379:methods
D002232:Carbocyanines; D011506:Proteins; C089187:cyanine dye 3; D049452:Green Fluorescent Proteins