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  • Title: Gaussian FRET two-hybrid assays for determining the stoichiometry of hetero-oligomeric complexes in single living cells.
    Author: Qu W, Du M, Yang F, Mai Z, Zhang C, Lin F, Ma Y, Chen T.
    Journal: Biochem Biophys Res Commun; 2019 May 07; 512(3):492-497. PubMed ID: 30904160.
    Abstract:
    Here we integrate multiple Gaussian-functions analysis into fluorescence resonance energy transfer (FRET) two-hybrid assays (Gaussian FRET two-hybrid assay) to determine the stoichiometric ratios of intracellular hetero-oligomers in single living cells. This method adopts in multiple Gaussian-functions to fit the E-count histograms of both donor- and acceptor-centric FRET efficiency (ED and EA) images of a single cell for obtaining the peak values (EDi and EAi), thus yielding the corresponding stoichiometric ratios (EDi/EAi) of intracellular hetero-oligomers. We performed Gaussian FRET two-hybrid assay for living Hela cells coexpressing different FRET tandem plasmids, and obtained consistent results with the expected values. Gaussian FRET two-hybrid assay for cells coexpressing Bad-CFP and Bcl-XL-YFP reveals that Bcl-XL binds with Bad to form a hetero-oligomeric complex with a stoichiometry of 2:1 on mitochondria.
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