Home List of Titles Dual-channel photobleaching FRET microscopy for improved resolution of protein association states in living cells
Please use this identifier to cite or link to this item: http://hdl.handle.net/1959.3/94145
- Dual-channel photobleaching FRET microscopy for improved resolution of protein association states in living cells
- Clayton, Andrew H. A.; Klonis, Nectarios; Cody, Stephen H.; Nice, Edouard C.
- Fluorescence resonance energy transfer (FRET) from a donor-labelled molecule to an acceptor-labelled molecule is a useful, proximity-based fluorescence tool to discriminate molecular states on the surface and in the interior of cells. Most microscope-based determinations of FRET yield only a single value, the interpretation of which is necessarily model-dependent. In this paper we demonstrate two new measurements of FRET heterogeneity using selective donor photobleaching in combination with synchronous donor/acceptor detection based on either (1) full kinetic analysis of donor-detected and acceptor-detected donor photobleaching or (2) a simple time-based ratiometric approach. We apply the new methods to study the cell surface distribution of concanavalin A yielding estimates of FRET and non-FRET population distributions, as well as FRET efficiencies within the FRET populations.
- Publication type
- Journal article
- European Biophysics Journal, Vol. 34, no. 1 (Feb 2005), pp. 82-90
- Publication year
- FOR Code(s)
- 0299 Other Physical Sciences
- Fluorescein; Fluorescence resonance energy transfer; FRET; Oligomerisation; Protein interactions; Rhodamine; Two-component analysis
- Publisher URL
- Copyright © EBSA 2004.
- Peer reviewed