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利用体外荧光共振能量转移研究毫秒时间尺度蛋白质配合物的动力学

JoVE Journal
Bioquímica

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JoVE Journal Bioquímica

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

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利用体外荧光共振能量转移研究毫秒时间尺度蛋白质配合物的动力学

DOI:

10.3791/59038-v

•

10:50 min

•

March 14, 2019

•

Melaku Garsamo³, Yun Zhou³, Xing Liu³

¹Department of Biochemistry,Purdue University, ²Department of Botany and Plant Pathology,Purdue University, ³Center for Plant Biology,Purdue University

Capítulos

00:04Título
00:33Designing the FRET Assay
01:12Preparation of Cul1^AMC•Rbx1, the FRET Donor Protein
04:42Preparation of ^FlAsHCand1, the FRET Acceptor Protein
05:18Testing and Confirmation of the FRET Assay
06:08Measuring the Association Rate Constant
07:37Measuring the Dissociation Rate Constant of Cul1•Cand1 in the Presence of Skp•F-box Protein
08:33Results: Dynamics Of Protein Complexes
10:30Conclusion

Summary

Tadução automática

蛋白质-蛋白质相互作用对生物系统至关重要, 对结合动力学的研究为蛋白质复合物的动力学和功能提供了见解。我们描述了一种利用荧光共振能量转移和停止流动技术量化蛋白质复合物动力学参数的方法。

Tags

Keywords: FRET Protein Complex Kinetics Association Dissociation COL1 CAND1 7-amino-4-methylcoumarin FlASH Cell Lysis Sonication Centrifugation Glutathione Beads Elution Thrombin Buffer A

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