Characterizing Protein‐Protein Interactions by Sedimentation Velocity Analytical Ultracentrifugation

Patrick H. Brown1, Andrea Balbo1, Peter Schuck1

1 National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 18.15
DOI:  10.1002/0471142735.im1815s81
Online Posting Date:  May, 2008
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Abstract

This unit introduces the basic principles and practice of sedimentation velocity analytical ultracentrifugation for the study of reversible protein interactions, such as the characterization of self‐association, heterogeneous association, multi‐protein complexes, binding stoichiometry, and the determination of association constants. The analytical tools described include sedimentation coefficient and molar mass distributions, multi‐signal sedimentation coefficient distributions, Gilbert‐Jenkins theory, different forms of isotherms, and global Lamm equation modeling. Concepts for the experimental design are discussed, and a detailed step‐by‐step protocol guiding the reader through the experiment and the data analysis is available as an Internet resource. Curr. Protoc. Immunol. 81:18.15.1‐18.15.39. © 2008 by John Wiley & Sons, Inc.

Keywords: sedimentation equilibrium; sedimentation velocity; chemical equilibria; reversible interactions; multi‐protein complex; analytical ultracentrifugation; size‐distribution; Gilbert‐Jenkins theory; Lamm equation; Bayesian analysis

     
 
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Table of Contents

  • Introduction
  • Basic Principles: Theory
  • Basic Principles: Experimental
  • Complementarity to Other Biophysical Techniques
  • Summary
  • Literature Cited
  • Figures
     
 
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Materials

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Figures

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Literature Cited

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Key References
Internet Resources
   http://www.analyticalultracentrifugation.com/SVProtocols.htm
  Step‐by‐step protocol for SV experiments (A. Balbo, P. Brown, P. Schuck).
   http://www.analyticalultracentrifugation.com
  Homepage of SEDFIT with help resources, including tutorials, step‐by‐step examples, complete function reference, and literature references.
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  Homepage of SEDPHAT with help resources, including tutorials, complete function reference, and literature references.
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