Determination of Membrane Protein Molecular Weight Using Sedimentation Equilibrium Analytical Ultracentrifugation

Karen G. Fleming1

1 Thomas C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, Maryland
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 7.12
DOI:  10.1002/0471140864.ps0712s53
Online Posting Date:  August, 2008
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Abstract

Both the stoichiometry and stability of native membrane protein complexes pose challenges to understanding the biology of these proteins. Sedimentation equilibrium analytical ultracentrifugation is well recognized as a thermodynamically rigorous technique for determining these quantities for macromolecules. This unit describes the experimental strategies that can be used to extract this information for membrane proteins reconstituted in vitro in detergent micelle or detergent/lipid mixed micelle solutions. Curr. Protoc. Protein Sci. 53:7.12.1‐7.12.13. © 2008 by John Wiley & Sons, Inc.

Keywords: membrane protein; thermodynamics; analytical ultracentrifugation

     
 
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Table of Contents

  • Introduction
  • Theory
  • Strategic Planning
  • Basic Protocol 1: Sedimentation Equilibrium to Determine Molecular Weight of Membrane Proteins
  • Conclusion
  • Acknowledgement
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Sedimentation Equilibrium to Determine Molecular Weight of Membrane Proteins

  Materials
  • Proteins of interest reconstituted in detergent of interest
  • Detergent micelle solution at appropriate concentration (for reference samples)
  • Heavy water (2H 2O, deuterium oxide; Sigma)
  • Sedimentation equilibrium cells (standard six‐sector, charcoal‐filled, epon cells recommended; Beckman)
  • Analytical ultracentrifuge with absorbance optics and quartz windows (Beckman XL‐A or XL‐I): for membrane protein analysis
  • Analytical ultracentrifuge with interference optics and sapphire windows (Beckman XL‐I): for determining matching density in detergent
  • Software program (e.g., WinMatch; available at http://www.bbri.org/rasmb) to determine if equilibrium is reached
  • Data trimming programs (e.g., WinReedit or Sedanal, available at http://www.bbri.org/rasmb)
  • Nonlinear least squares analysis program for analyzing sedimentation equilibrium data (e.g., NONLIN; available at http://www.bbri.org/rasmb)
  • Additional reagents and equipment for equilibrating protein in detergent micelle solution (unit 4.8)
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Figures

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Literature Cited

Literature Cited
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