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Quantitative fluorescence resonance energy transfer (FRET) measurement with acceptor photobleaching and spectral unmixing.
- Source :
- Journal of Microscopy; Aug2004, Vol. 215 Issue 2, p162-173, 12p
- Publication Year :
- 2004
-
Abstract
- Fluorescence resonance energy transfer (FRET) by acceptor photobleaching is a simple but effective tool for measurements of protein–protein interactions. Until recently, it has been restricted to qualitative or relative assessments owing to the spectral bleed-through contamination resulting from fluorescence overlap between the donor and the acceptor. In this paper, we report a quantitative algorithm that combines the spectral unmixing technique with FRET by acceptor photobleaching. By spectrally unmixing the emissions before and after photobleaching, it is possible to resolve the spectral bleed-through and retrieve the FRET efficiency/interaction distance quantitatively. Using a human keratinocyte cell line transfected with cyan fluorescent protein (CFP)- and yellow fluorescent protein (YFP)-tagged Cx26 connexins as an example, FRET information at homotypic gap junctions is measured and compared with well-established methods. Results indicate that the new approach is sensitive, flexible, instrument independent and solely FRET dependent. It can achieve FRET estimations similar to that from a sensitized emission FRET method. This approach has a great advantage in providing the relative concentrations of the donor and the acceptor; this is, for example, very important in the comparative study of cell populations with variable expression levels. [ABSTRACT FROM AUTHOR]
- Subjects :
- ENERGY transfer
FORCE & energy
ENERGY storage
RESONANCE
PROTEINS
BIOMOLECULES
Subjects
Details
- Language :
- English
- ISSN :
- 00222720
- Volume :
- 215
- Issue :
- 2
- Database :
- Complementary Index
- Journal :
- Journal of Microscopy
- Publication Type :
- Academic Journal
- Accession number :
- 13801575
- Full Text :
- https://doi.org/10.1111/j.0022-2720.2004.01365.x