Chapter 9
Sedimentation Velocity Methods for the Characterization
of Protein Heterogeneity and Protein Affinity Interactions
Christine Ebel and Catherine Birck
Abstract
Sedimentation velocity analytical ultracentrifugation is a powerful and versatile tool for the characterization
of proteins and macromolecular complexes in solution. The direct modeling of the sedimentation process
using modern computational strategies allows among others to assess the homogeneity/heterogeneity state
of protein samples and to characterize protein associations. In this chapter, we will provide theoretical
backgrounds and protocols to analyze the size distribution of protein samples and to determine the affinity
of protein–protein hetero-associations.
Key words Sedimentation velocity, Analytical ultracentrifugation, Protein heterogeneity, Sedimentation coefficient, Hetero-association, Protein interaction
1 Introduction
The characterization of biological samples to assess their homogeneity and to study the formation of protein complexes is of interest
in many areas of biological research. Getting a homogeneous sample is among others an essential requirement for the accuracy and
reproducibility of the experimental results, the correct interpretation of biophysical data, and high-resolution structural work. It will
also benefit protein interaction studies, as the presence of aggregates may decrease the fraction of protein competent for binding or
lead to improper modeling of the interaction. Few methods allow
the characterization of protein homogeneity and protein binding
properties. It is the case of sedimentation velocity (SV) analytical
ultracentrifugation (AUC) which is a powerful biophysical technique commonly used to determine the size and shape of macromolecules in solution and to study protein interactions. It is an
absolute method based on first principles that benefits from constant developments in analysis software. In SV, macromolecules
loaded in sector-shaped cells are separated at high centrifugal
force and their sedimentation profiles, the evolution of the protein
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_9, © Springer Science+Business Media, LLC, part of Springer Nature 2021
155
Sedimentation Velocity Methods for the Characterization
of Protein Heterogeneity and Protein Affinity Interactions
Christine Ebel and Catherine Birck
Abstract
Sedimentation velocity analytical ultracentrifugation is a powerful and versatile tool for the characterization
of proteins and macromolecular complexes in solution. The direct modeling of the sedimentation process
using modern computational strategies allows among others to assess the homogeneity/heterogeneity state
of protein samples and to characterize protein associations. In this chapter, we will provide theoretical
backgrounds and protocols to analyze the size distribution of protein samples and to determine the affinity
of protein–protein hetero-associations.
Key words Sedimentation velocity, Analytical ultracentrifugation, Protein heterogeneity, Sedimentation coefficient, Hetero-association, Protein interaction
1 Introduction
The characterization of biological samples to assess their homogeneity and to study the formation of protein complexes is of interest
in many areas of biological research. Getting a homogeneous sample is among others an essential requirement for the accuracy and
reproducibility of the experimental results, the correct interpretation of biophysical data, and high-resolution structural work. It will
also benefit protein interaction studies, as the presence of aggregates may decrease the fraction of protein competent for binding or
lead to improper modeling of the interaction. Few methods allow
the characterization of protein homogeneity and protein binding
properties. It is the case of sedimentation velocity (SV) analytical
ultracentrifugation (AUC) which is a powerful biophysical technique commonly used to determine the size and shape of macromolecules in solution and to study protein interactions. It is an
absolute method based on first principles that benefits from constant developments in analysis software. In SV, macromolecules
loaded in sector-shaped cells are separated at high centrifugal
force and their sedimentation profiles, the evolution of the protein
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_9, © Springer Science+Business Media, LLC, part of Springer Nature 2021
155