One‐Dimensional Electrophoresis Using Nondenaturing Conditions

Sean R. Gallagher1

1 Motorola Corporation BioChip Systems, Tempe, Arizona
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 6.5
DOI:  10.1002/0471143030.cb0605s05
Online Posting Date:  May, 2001
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Abstract

Two protocols for nondenaturing or native gel electrophoresis (i.e., in the absence of denaturing agents, detergent, or urea) are presented in this unit and offer a useful technique for analyzing the native size of proteins, subunit structure, and optimal separation. Mobility of proteins in native gels depends on size, shape, and intrinsic charge. Continuous PAGE is very flexible and permits cationic and anionic separations over a broad range of pH. Discontinuous PAGE is limited to separations of proteins that are negatively charged at neutral pH.

     
 
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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
  • 4× acetic acid gel buffer (200 mM acetic acid, pH 3.7 to 5.6; see recipe)
  • 4× phosphate gel buffer (400 mM sodium phosphate, pH 5.8 to 8.0; see recipe)
  • 4× Tris gel buffer (200 mM Tris⋅Cl, pH 7.1 to 8.9; see recipe)
  • 4× 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 6.1) and staining proteins in gels ( appendix 3A)

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 2A)
  • 4× Tris⋅Cl, pH 6.8 (0.5 M Tris⋅Cl)
  • Protein sample of interest
  • 2× Tris/glycerol sample buffer (see recipe)
  • Native protein standards (e.g., Sigma nondenatured protein molecular weight kit)
  • Tris/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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