Quantitative Analysis of Protein Self-Association by Sedimentation Velocity.

Szczegóły
Abstrakt

Tytuł:: Quantitative Analysis of Protein Self-Association by Sedimentation Velocity.
Autorzy:: Zhao H; Dynamics of Macromolecular Assembly Section, Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland.
Li W; National Protein Science Facility, School of Life Science, Tsinghua University, Beijing, China.
Chu W; National Protein Science Facility, School of Life Science, Tsinghua University, Beijing, China.
Bollard M; Dynamics of Macromolecular Assembly Section, Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland.
Adão R; Dynamics of Macromolecular Assembly Section, Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland.
Schuck P; Dynamics of Macromolecular Assembly Section, Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland.
Źródło:: Current protocols in protein science [Curr Protoc Protein Sci] 2020 Sep; Vol. 101 (1), pp. e109.
Typ publikacji:: Journal Article; Research Support, N.I.H., Intramural
Język:: English
MeSH Terms:: Chemical Fractionation/*instrumentation
Proteins/*isolation & purification
Ultracentrifugation/*standards
Buffers ; Calibration ; Humans ; Hydrodynamics ; Molecular Weight ; Proteins/chemistry ; Temperature ; Ultracentrifugation/instrumentation
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Grant Information:: Z01 EB000051 United States ImNIH Intramural NIH HHS
Contributed Indexing:: Keywords: binding isotherm; instrument calibration; protein self-association; sedimentation velocity
Substance Nomenclature:: 0 (Buffers)
0 (Proteins)
Entry Date(s):: Date Created: 20200703 Date Completed: 20210106 Latest Revision: 20221005
Update Code:: 20240105
PubMed Central ID:: PMC7430532
DOI:: 10.1002/cpps.109
PMID:: 32614509
: Czasopismo naukowe

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.
(©2020 Wiley Periodicals LLC. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

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