FRET-based Stoichiometry Measurements of Protein Complexes in vitro

被引：0
|
作者：
Mattiroli, Francesca [1 ]
Gu, Yajie [1 ,2 ]
Luger, Karolin [1 ]
机构：
[1] Univ Colorado, Howard Hughes Med Inst, Dept Chem & Biochem, Boulder, CO 80309 USA
[2] Colorado State Univ, Dept Biochem & Mol Biol, Ft Collins, CO 80523 USA
来源：
BIO-PROTOCOL | 2018年 / 8卷 / 03期
关键词：
Stoichiometry; FRET; Histones; Fluorescence; Complex formation; Job plot;
D O I：
10.21769/BioProtoc.2713
中图分类号：
Q [生物科学];
学科分类号：
07 ; 0710 ; 09 ;
摘要：
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.
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页数：7
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