Open AccessExEm-FRET two-hybrid assay: FRET two-hybrid assay based on linear unmixing of excitation-emission spectra
Author(s) -
Chenshuang Zhang,
Yangpei Liu,
Wenfeng Qu,
Wenhua Su,
Mingde Du,
Fangfang Yang,
Tongsheng Chen
Publication year - 2019
Publication title -
optics express
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.394
H-Index - 271
ISSN - 1094-4087
DOI - 10.1364/oe.27.018282
Subject(s) - förster resonance energy transfer , acceptor , fluorescence , excitation , spectral line , analytical chemistry (journal) , materials science , chemistry , optics , physics , chromatography , quantum mechanics , astronomy , condensed matter physics
Simultaneous linear unmixing of excitation-emission spectra (ExEm-unmixing)-based fluorescence resonance energy transfer (FRET) two-hybrid assay method, named as ExEm-FRET two-hybrid assay, was developed for evaluating the stoichiometric ratio of macromolecular complexes in living cells. Linear unmixing of the excitation-emission spectra (SDA) of cells obtains the weight factors of donor (WD), acceptor (WA) and acceptor sensitization (WS), yielding ED and EA (donor- and acceptor-centric FRET efficiency) images. ExEm-FRET two-hybrid assay employs pixel-to-pixel titration curves of ED/EA versus the free acceptor (Ca)/donor (Cd) concentration deduced from the three weight factors to obtain EA,max and ED,max (the maximal EA and ED), thus yielding the stoichiometric ratio (EA,max/ED,max) of donor-tagged protein to acceptor-tagged protein.