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Titolo:
Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy
Autore:
Kenworthy, AK;
Indirizzi:
Johns Hopkins Univ, Dept Biol, Baltimore, MD 21218 USA Johns Hopkins UnivBaltimore MD USA 21218 t Biol, Baltimore, MD 21218 USA
Titolo Testata:
METHODS
fascicolo: 3, volume: 24, anno: 2001,
pagine: 289 - 296
SICI:
1046-2023(200107)24:3<289:IPIUFR>2.0.ZU;2-Q
Fonte:
ISI
Lingua:
ENG
Soggetto:
GPI-ANCHORED PROTEINS; CELL-SURFACE; LIVING CELLS; PHOTOBLEACHING KINETICS; FRET MICROSCOPY; APICAL SURFACE; CHOLERA-TOXIN; 2 DIMENSIONS; CYCLIC-AMP; MDCK CELLS;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
56
Recensione:
Indirizzi per estratti:
Indirizzo: Kenworthy, AK NICHD, Cell Biol & Metab Branch, NIH, Bldg 18T,Room 101, Bethesda, MD 20892 USA NICHD Bldg 18T,Room 101 Bethesda MD USA 20892 , MD 20892 USA
Citazione:
A.K. Kenworthy, "Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy", METHODS, 24(3), 2001, pp. 289-296

Abstract

Fluorescence resonance energy transfer (FRFT) detects the proximity of fluorescently labeled molecules over distances > 100 Angstrom. When performed in a fluorescence microscope, FRET can be used to map protein-protein interactions in vivo. We here describe a FRET microscopy method that can be usedto determine whether proteins that are colocalized at the level of light microscopy interact with one another. This method can be implemented using digital microscopy systems such as a confocal microscope or a wide-field fluorescence microscope coupled to a charge-coupled device (CCD) camera. It isreadily applied to samples prepared with standard immunofluorescence techniques using antibodies labeled with fluorescent dyes that act as a donor and acceptor pair for FRET. Energy transfer efficiencies are quantified basedon the release of quenching of donor fluorescence due to FRET, measured bycomparing the intensity of donor fluorescence before and after complete photobleaching of the acceptor. As described, this method uses Cy3 and Cy5 asthe donor and acceptor fluorophores, but can be adapted for other FRET pairs Including cyan fluorescent protein and yellow fluorescent protein, (C) 2001 Academic Press.

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Documento generato il 30/11/20 alle ore 16:43:49