ExEm-FRET two-hybrid assay: FRET two-hybrid assay based on linear unmixing of excitation-emission spectra | Zendy

Chenshuang Zhang | Zendy; Yangpei Liu | Zendy; Wenfeng Qu | Zendy; Wenhua Su | Zendy; Mengyan Du | Zendy; Fangfang Yang | Zendy; Tongsheng Chen | Zendy

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ExEm-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,

Mengyan 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) , stoichiometry , emission spectrum , 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.

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