Quantitative Analysis of Protein Self-Association by Sedimentation Velocity

Q1 Biochemistry, Genetics and Molecular Biology

Current Protocols in Protein Science Pub Date : 2020-07-02 DOI:10.1002/cpps.109

Huaying Zhao, Wenqi Li, Wendan Chu, Mary Bollard, Regina Adão, Peter Schuck

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引用次数: 6

Abstract

Sedimentation velocity analytical ultracentrifugation is a powerful classical method to study protein self-association processes in solution based on the size-dependent macromolecular migration in the centrifugal field. This technique can elucidate the assembly scheme, measure affinities ranging from picomolar to millimolar K_d, and in favorable cases provide information on oligomer lifetimes and hydrodynamic shape. The present step-by-step protocols detail the essential steps of instrument calibration, experimental setup, and data analysis. Using a widely available commercial protein as a model system, the protocols invite replication and comparison with our results. A commentary discusses principles for modifications in the protocols that may be necessary to optimize application of sedimentation velocity analysis to other self-associating proteins. ©2020 Wiley Periodicals LLC.

Basic Protocol 1: Measurement of external calibration factors

Basic Protocol 2: Sedimentation velocity experiment for protein self-association

Basic Protocol 3: Sedimentation coefficient distribution analysis in SEDFIT and isotherm analysis in SEDPHAT

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用沉降速度定量分析蛋白质的自结合

沉降速度分析超离心是一种基于离心场中大小依赖的大分子迁移来研究蛋白质在溶液中自结合过程的有力的经典方法。该技术可以阐明组装方案，测量从皮摩尔到毫摩尔Kd的亲和度，并在有利的情况下提供有关低聚物寿命和流体动力学形状的信息。目前一步一步的协议详细说明仪器校准，实验设置和数据分析的基本步骤。使用广泛可用的商业蛋白质作为模型系统，该方案可以与我们的结果进行复制和比较。评论讨论了可能需要优化沉降速度分析在其他自结合蛋白中的应用的协议修改原则。©2020 Wiley期刊公司基本方案1:外部校准因子的测量基本方案2:蛋白质自结合的沉降速度实验基本方案3:SEDFIT中的沉降系数分布分析和SEDPHAT中的等温线分析

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来源期刊

Current Protocols in Protein Science Biochemistry, Genetics and Molecular Biology-Biochemistry

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期刊介绍： With the mapping of the human genome, more and more researchers are exploring protein structures and functions in living organisms. Current Protocols in Protein Science provides protein scientists, biochemists, molecular biologists, geneticists, and others with the first comprehensive suite of protocols for this growing field.