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{"title":"用沉降速度定量分析蛋白质的自结合","authors":"Huaying Zhao, Wenqi Li, Wendan Chu, Mary Bollard, Regina Adão, Peter Schuck","doi":"10.1002/cpps.109","DOIUrl":null,"url":null,"abstract":"<p>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 <i>K</i><sub>d</sub>, 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.</p><p><b>Basic Protocol 1</b>: Measurement of external calibration factors</p><p><b>Basic Protocol 2</b>: Sedimentation velocity experiment for protein self-association</p><p><b>Basic Protocol 3</b>: Sedimentation coefficient distribution analysis in SEDFIT and isotherm analysis in SEDPHAT</p>","PeriodicalId":10866,"journal":{"name":"Current Protocols in Protein Science","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2020-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpps.109","citationCount":"6","resultStr":"{\"title\":\"Quantitative Analysis of Protein Self-Association by Sedimentation Velocity\",\"authors\":\"Huaying Zhao, Wenqi Li, Wendan Chu, Mary Bollard, Regina Adão, Peter Schuck\",\"doi\":\"10.1002/cpps.109\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>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 <i>K</i><sub>d</sub>, 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.</p><p><b>Basic Protocol 1</b>: Measurement of external calibration factors</p><p><b>Basic Protocol 2</b>: Sedimentation velocity experiment for protein self-association</p><p><b>Basic Protocol 3</b>: Sedimentation coefficient distribution analysis in SEDFIT and isotherm analysis in SEDPHAT</p>\",\"PeriodicalId\":10866,\"journal\":{\"name\":\"Current Protocols in Protein Science\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2020-07-02\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.1002/cpps.109\",\"citationCount\":\"6\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Current Protocols in Protein Science\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1002/cpps.109\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"Biochemistry, Genetics and Molecular Biology\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current Protocols in Protein Science","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cpps.109","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"Biochemistry, Genetics and Molecular Biology","Score":null,"Total":0}
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