Please use this identifier to cite or link to this item: http://hdl.handle.net/1959.3/94145

Title

Dual-channel photobleaching FRET microscopy for improved resolution of protein association states in living cells

Author(s)

Clayton, Andrew H. A.;  Klonis, Nectarios;  Cody, Stephen H.;  Nice, Edouard C.

Abstract

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

Source

European Biophysics Journal, Vol. 34, no. 1 (Feb 2005), pp. 82-90

Publication year

2005

FOR Code(s)

0299 Other Physical Sciences

Keyword(s)

Fluorescein;  Fluorescence resonance energy transfer;  FRET;  Oligomerisation;  Protein interactions;  Rhodamine;  Two-component analysis

Publisher

Springer

ISSN

0175-7571

Publisher URL

http://dx.doi.org/10.1007/s00249-004-0427-y

Copyright

Copyright © EBSA 2004.

Peer reviewed