ExEm-FRET two-hybrid assay: FRET two-hybrid assay based on linear unmixing of excitation-emission spectra
| Autor: | Fangfang Yang, Chenshuang Zhang, Wenhua Su, Yangpei Liu, Wenfeng Qu, Tongsheng Chen, Mengyan Du |
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| Rok vydání: | 2019 |
| Předmět: |
Titration curve
business.industry Chemistry Two-hybrid screening Analytical chemistry 02 engineering and technology 021001 nanoscience & nanotechnology 01 natural sciences Acceptor Fluorescence Atomic and Molecular Physics and Optics 010309 optics Förster resonance energy transfer Optics 0103 physical sciences Molecule Emission spectrum 0210 nano-technology business Excitation |
| Zdroj: | Optics express. 27(13) |
| ISSN: | 1094-4087 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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