title: TripleFRET measurements in flow cytometry.
abstract: A frequently used method for viewing protein interactions and conformation, Förster (fluorescence) resonance energy transfer (FRET), has traditionally been restricted to two fluorophores. Lately, several methods have been introduced to expand FRET methods to three species. We present a method that allows the determination of FRET efficiency in three-dye systems on a flow cytometer. TripleFRET accurately reproduces energy transfer efficiency values measured in two-dye systems, and it can indicate the presence of trimeric complexes, which is not possible with conventional FRET methods. We also discuss the interpretation of energy transfer values obtained with tripleFRET in relation to spatial distribution of labeled molecules, specifically addressing the limitations of using total energy transfer to determine molecular distance.
authors.affiliation: Department of Biophysics and Cell Biology, Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, Hungary.
authors.forename: Ákos
authors.initials: Á
authors.lastname: Fábián
authors.forename: Gábor
authors.initials: G
authors.lastname: Horváth
authors.forename: György
authors.initials: G
authors.lastname: Vámosi
authors.forename: György
authors.initials: G
authors.lastname: Vereb
authors.forename: János
authors.initials: J
authors.lastname: Szöllősi
mesh_terms: D000906:Antibodies / Q000737:chemistry* / Q000276:immunology; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry / Q000276:immunology; D002460:Cell Line; D005434:Flow Cytometry / Q000379:methods / Q000706:statistics & numerical data*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods / Q000706:statistics & numerical data*; D005456:Fluorescent Dyes / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry / Q000276:immunology; D011485:Protein Binding; D013194:Staining and Labeling; D000068878:Trastuzumab
chemical_list: D000906:Antibodies; D061067:Antibodies, Monoclonal, Humanized; D005456:Fluorescent Dyes; D018796:Immunoconjugates; C485206:pertuzumab; D000068878:Trastuzumab