Quantitative Analysis of Protein Self‐Association by Sedimentation Velocity | Zendy

Zhao Huaying | Zendy; Li Wenqi | Zendy; Chu Wendan | Zendy; Bollard Mary | Zendy; Adão Regina | Zendy; Schuck Peter | Zendy

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Quantitative Analysis of Protein Self‐Association by Sedimentation Velocity

Author(s) -

Zhao Huaying,

Li Wenqi,

Chu Wendan,

Bollard Mary,

Adão Regina,

Schuck Peter

Publication year - 2020

Publication title -

current protocols in protein science

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 1.409

H-Index - 32

eISSN - 1934-3663

pISSN - 1934-3655

DOI - 10.1002/cpps.109

Subject(s) - analytical ultracentrifugation , sedimentation , ultracentrifuge , sedimentation coefficient , calibration , protocol (science) , replication (statistics) , sedimentation equilibrium , biological system , measure (data warehouse) , chemistry , computer science , chromatography , analytical chemistry (journal) , data mining , statistics , biology , mathematics , biochemistry , medicine , paleontology , alternative medicine , pathology , sediment , enzyme

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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