Open AccessFRET-based Stoichiometry Measurements of Protein Complexes in vitro
Author(s) -
Francesca Mattiroli,
Yajie Gu,
Karolin Luger
Publication year - 2018
Publication title -
bio-protocol
Language(s) - English
Resource type - Journals
ISSN - 2331-8325
DOI - 10.21769/bioprotoc.2713
Subject(s) - förster resonance energy transfer , stoichiometry , chemistry , fluorescence , analytical chemistry (journal) , resonance (particle physics) , in vitro , biophysics , crystallography , chromatography , biochemistry , biology , physics , quantum mechanics , particle physics
For a complete understanding of biochemical reactions, information on complex stoichiometry is essential. However, measuring stoichiometry is experimentally challenging. Our lab has developed a FRET-based assay to study protein complex stoichiometry in vitro . This assay, also known as Job plot, is set up as a continuous variation of the molar ratio between the two species, kept at constant total concentration. The FRET (Fluorescence Resonance Energy Transfer) between the two fluorescently-labeled proteins is measured and the stoichiometry is inferred from the sample with highest FRET signal. This approach allows us to assess complex stoichiometry in solution.