Overview of Current Methods in Sedimentation Velocity and Sedimentation Equilibrium Analytical Ultracentrifugation

Huaying Zhao1, Chad A. Brautigam2, Rodolfo Ghirlando3, Peter Schuck1

1 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, 2 Department of Biochemistry, The University of Texas Southwestern Medical Center, Dallas, Texas, 3 Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 20.12
DOI:  10.1002/0471140864.ps2012s71
Online Posting Date:  February, 2013
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Modern computational strategies have allowed for the direct modeling of the sedimentation process of heterogeneous mixtures, resulting in sedimentation velocity (SV) size‐distribution analyses with significantly improved detection limits and strongly enhanced resolution. These advances have transformed the practice of SV, rendering it the primary method of choice for most existing applications of analytical ultracentrifugation (AUC), such as the study of protein self‐ and hetero‐association, the study of membrane proteins, and applications in biotechnology. New global multisignal modeling and mass conservation approaches in SV and sedimentation equilibrium (SE), in conjunction with the effective‐particle framework for interpreting the sedimentation boundary structure of interacting systems, as well as tools for explicit modeling of the reaction/diffusion/sedimentation equations to experimental data, have led to more robust and more powerful strategies for the study of reversible protein interactions and multiprotein complexes. Furthermore, modern mathematical modeling capabilities have allowed for a detailed description of many experimental aspects of the acquired data, thus enabling novel experimental opportunities, with important implications for both sample preparation and data acquisition. The goal of the current unit is to describe the current tools for the study of soluble proteins, detergent‐solubilized membrane proteins and their interactions by SV and SE. Curr. Protoc. Protein Sci. 71:20.12.1‐20.12.49. © 2013 by John Wiley & Sons, Inc.

Keywords: sedimentation equilibrium; sedimentation velocity; chemical equilibria; reversible interaction; size‐distribution; multiprotein complex; analytical ultracentrifugation; protein hydrodynamics

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Table of Contents

  • Introduction
  • Basic Principles
  • Experimental Design and Protocols
  • Data Analysis
  • Acknowledgements
  • Literature Cited
  • Figures
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Internet Resources
   There are abundant resources available on the Internet covering software, detailed experimental step‐by‐step protocols, video instructions, and discussion forums. The following is only a small subset facilitating the application of the methods described in the present unit.
  General practical tools to help designing AUC experiments, including detailed step‐by‐step protocols for conducting SE and SV experiments, a table with criteria for buffer selection, a grid for SV run conditions with different optical systems, instructions for radial calibration of the interference system, and a tutorial for the test for equilibrium.
  A video showing the assembly of cells (Balbo et al., ).
  SEDFIT and SEDPHAT can be freely downloaded from these sites. All methods discussed above are implemented in SEDFIT and SEDPHAT, unless otherwise stated.
  Extensive online help system and documentation, including a tutorial for ‘getting started’ in SEDFIT
  Step‐by‐step examples for the data analysis with c(s).
  Getting started’ tools for SEDPHAT.
  Tutorials on special topics of AUC and/or SEDFIT or SEDPHAT analyses.
  Movies illustrating the mechanism of migration of the reaction boundary.
  A step‐by‐step protocol for sedimentation equilibrium analyses.
  GUSSI download site.
  SEDNTERP for the calculation of buffer densities and viscosities, as well as protein extinction coefficients and partial specific volumes, at John Philo's Web site.
  SEDFIT‐L discussion forum.
  SEDPHAT‐L discussion forum.
  RASMB discussion forum.
  These two Web sites provide current information on upcoming workshops.
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