One‐Dimensional Electrophoresis Using Nondenaturing Conditions

Sean R. Gallagher1

1 Hoefer Pharmacia Biotech, San Francisco
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 10.3
DOI:  10.1002/0471140864.ps1003s00
Online Posting Date:  May, 2001
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Abstract

Nondenaturing or “native“ electrophoresis—i.e., electrophoresis in the absence of denaturants such as detergents and urea—is an often‐overlooked technique for determining the native size, subunit structure, and optimal separation of a protein. Two protocols are presented in this unit: continuous PAGE, which is highly flexible, permitting cationic and anionic electrophoresis over a full range of pH, and discontinuous PAGE, which is limited to proteins negatively charged at neutral pH but provides high resolution for accurate size calibration.

     
 
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Table of Contents

  • Basic Protocol 1: Continuous Electrophoresis in Nondenaturing Polyacrylamide Gels
  • Alternate Protocol 1: Native Discontinuous Electrophoresis and Generation of Molecular Weight Standard Curves (Ferguson Plots)
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Continuous Electrophoresis in Nondenaturing Polyacrylamide Gels

  Materials
  • recipe4× acetic acid gel buffer (200 mM acetic acid, pH 3.7 to 5.6; see recipe)
  • recipe4× phosphate gel buffer (400 mM sodium phosphate, pH 5.8 to 8.0; see recipe)
  • recipe4× Tris gel buffer (200 mM Tris⋅Cl, pH 7.1 to 8.9; see recipe)
  • recipe4× glycine gel buffer (200 mM glycine, pH 8.6 to 10.6; see recipe)
  • 300 mM sodium sulfite (0.38 g in 10 ml H 2O; used in acetic acid gel preparation)
  • Protein samples to be analyzed
  • Native protein standards
  • Electrophoresis buffer: appropriate 4× gel buffer diluted to 1× with H 2O
  • 75‐ml side‐arm flask (used in gel preparation)
  • Additional reagents and equipment for gel electrophoresis (unit 10.1) and staining proteins in gels (unit 10.5)

Alternate Protocol 1: Native Discontinuous Electrophoresis and Generation of Molecular Weight Standard Curves (Ferguson Plots)

  Materials
  • 4× Tris⋅Cl, pH 8.8 ( 1.5 M Tris⋅Cl; appendix 2E)
  • 4× Tris⋅Cl, pH 6.8 ( 0.5 M Tris⋅Cl; appendix 2E)
  • Protein sample of interest
  • recipe2× Tris/glycerol sample buffer (see recipe)
  • Native protein standards (e.g., Sigma nondenatured protein molecular weight kit)
  • recipeTris/glycine electrophoresis buffer (see recipe)
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Figures

Videos

Literature Cited

Literature Cited
   Andrews, A.T. 1986. Electrophoresis: Theory, Techniques and Biochemical and Clinical Applications, 2nd ed. Oxford University Press, New York.
   Ferguson, K.A. 1964. Starch‐gel electrophoresis—application to the classification of pituitary proteins and polypeptides. Metabolism 13:985‐1002.
   Hames, D. 1990. One‐dimensional polyacrylamide gel electrophoresis. In Gel Electrophoresis of Proteins: A Practical Approach, 2nd ed. (B.D. Hames and D. Rickwood, eds.) pp.1‐147. Oxford University Press New York.
   Hedrick, J.L. and Smith, A.J. 1968. Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis. Arch. Biochem. Biophys. 126:155‐164.
   Rodbard, D. and Chrambach, A. 1971. Estimation of molecular radius, free mobility, and valence using polyacrylamide gel electrophoresis. Anal. Biochem. 40:95‐134.
   Schägger, H. 1994. Native gel electrophoresis. In A Practical Guide to Membrane Protein Purification (G. Von Jagow and H. Schägger, eds.) pp.81‐104. Academic Press, San Diego.
   Sigma. 1986. Nondenatured protein molecular weight marker kit (Technical Bulletin No. MKR‐137). Sigma Chemical Company, St. Louis, Mo.
Key Reference
   Andrews, 1986. See above.
  Covers a variety of electrophoretic techniques, including nondenaturing electrophoresis and Ferguson plots.
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