Analysis of FRET-signals in the presence of free donors and acceptors Export

@article{citeulike:1805630, abstract = {A method for spectral analysis of FRET-signals is presented, taking into consideration both the contributions of unpaired donor and acceptor fluorophores and the influence of incomplete labeling of the interacting partners. It is shown that spectral analysis of intermolecular FRET cannot yield accurate values of the Forster energy transfer efficiency E, unless one of the interactors is in large excess and perfectly labeled. Instead, analysis of donor quenching yields a product of the form Efdpa, where fd is the fraction of donor-type molecules participating in donor-acceptor complexes and pa is the labeling probability of the acceptor. Similarly, analysis of sensitized emission yields a product involving Efa. The analysis of intramolecular FRET (e.g. of tandem constructs) yields the product Epa. We use our method to determine these values for a tandem construct of CFP and YFP and compare them with those obtained by standard acceptor photobleaching and fluoresecence lifetime measurements. We call the method lux-FRET, since it relies on linear unmixing of spectral components. 10.1529/biophysj.107.111773}, author = {Wlodarczyk, Jakub and Woehler, Andrew and Kobe, Fritz and Ponimaskin, Evgeni and Zeug, Andre and Neher, Erwin}, citeulike-article-id = {1805630}, citeulike-linkout-0 = {http://dx.doi.org/10.1529/biophysj.107.111773}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/17921223}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=17921223}, day = {5}, doi = {10.1529/biophysj.107.111773}, journal = {Biophys. J.}, keywords = {fret}, month = {October}, pages = {biophysj.107.111773+}, posted-at = {2008-03-13 10:33:06}, priority = {2}, title = {Analysis of FRET-signals in the presence of free donors and acceptors}, url = {http://dx.doi.org/10.1529/biophysj.107.111773}, year = {2007} }

TY - JOUR ID - citeulike:1805630 L3 - citeulike-article-id:1805630 N2 - A method for spectral analysis of FRET-signals is presented, taking into consideration both the contributions of unpaired donor and acceptor fluorophores and the influence of incomplete labeling of the interacting partners. It is shown that spectral analysis of intermolecular FRET cannot yield accurate values of the Forster energy transfer efficiency E, unless one of the interactors is in large excess and perfectly labeled. Instead, analysis of donor quenching yields a product of the form Efdpa, where fd is the fraction of donor-type molecules participating in donor-acceptor complexes and pa is the labeling probability of the acceptor. Similarly, analysis of sensitized emission yields a product involving Efa. The analysis of intramolecular FRET (e.g. of tandem constructs) yields the product Epa. We use our method to determine these values for a tandem construct of CFP and YFP and compare them with those obtained by standard acceptor photobleaching and fluoresecence lifetime measurements. We call the method lux-FRET, since it relies on linear unmixing of spectral components. 10.1529/biophysj.107.111773 JF - Biophys. J. TI - Analysis of FRET-signals in the presence of free donors and acceptors SP - biophysj.107.111773 KW - fret AU - Wlodarczyk, Jakub AU - Woehler, Andrew AU - Kobe, Fritz AU - Ponimaskin, Evgeni AU - Zeug, Andre AU - Neher, Erwin PY - 2007/10/05/ UR - http://dx.doi.org/10.1529/biophysj.107.111773 ER -