Linked Life Data

18638193

Source:http://linkedlifedata.com/resource/pubmed/id/18638193

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
Pt 1
pubmed:dateCreated
2008-7-21
pubmed:abstractText
To study protein-protein interactions by fluorescence energy transfer (FRET), the proteins of interest are tagged with either a donor or an acceptor fluorophore. For efficient FRET, fluorophores need to have a reasonable overlap of donor emission and acceptor excitation spectra. However, given the relatively small Stokes shift of conventional fluorescent proteins, donor and acceptor pairs with high FRET efficiencies have emission spectra that are difficult to separate. GFP and YFP are widely used in fluorescence microscopy studies. The spectral qualities of GFP and YFP make them one of the most efficient FRET donor-acceptor couples available. However, the emission peaks of GFP (510 nm) and YFP (527 nm) are spectrally too close for separation by conventional fluorescence microscopy. Difficulties in simultaneous detection of GFP and YFP with a fluorescence microscope are eliminated when spectral imaging and subsequent linear unmixing are applied. This allows FRET microscopy using these tags to study protein-protein interactions. We adapted the linear unmixing procedure from commercially available software (Zeiss) for use with acceptor photobleaching FRET using GFP and YFP as FRET pair. FRET efficiencies up to 52% for a GFP-YFP fusion protein were measured. To investigate the applicability of the procedure, we used two constituents of the nucleotide excision repair system, which removes UV-induced single-strand DNA damage. ERCC1 and XPF form a heterodimeric 5' endonuclease in nucleotide excision repair. FRET between ERCC1-GFP and XPF-YFP occurs with an efficiency of 30%.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jul
pubmed:issn
1365-2818
pubmed:author
pubmed:issnType
Electronic
pubmed:volume
231
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
97-104
pubmed:meshHeading
pubmed:year
2008
pubmed:articleTitle
Fluorescence resonance energy transfer of GFP and YFP by spectral imaging and quantitative acceptor photobleaching.
pubmed:affiliation
Department of Pathology, Josephine Nefkens Institute, Erasmus MC, PO Box 2040, 3000 CA Rotterdam, The Netherlands.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't