Transverse Urea‐Gradient Gel Electrophoresis

David P. Goldenberg1

1 University of Utah, Salt Lake City
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 7.4
DOI:  10.1002/0471140864.ps0704s03
Online Posting Date:  May, 2001
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Monitoring the cooperative unfolding transition induced when a protein is exposed to elevated temperature or a chemical denaturant is an important strategy for characterizing the conformational properties of a globular protein. This transition may be analyzed quantitatively by a variety of spectroscopic techniques, but a simpler alternative is described in this unit: urea‐gradient gel electrophoresis. The pattern produced in the resulting gel can be used to estimate both the free energy change for unfolding and the rate of the unfolding transition. In addition, the technique can help identify either covalent or conformational heterogeneity in a protein sample. Because urea‐gradient gel patterns are sensitive to several parameters, including hydrodynamic volume, net charge, and conformational stability, the technique can be particularly useful for comparing two forms of a protein, e.g., a natural form and the product of recombinant bacteria.

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • Gel overlay solution: 20% (v/v) ethanol/0.002% (w/v) bromophenol blue
  • recipeUrea/acrylamide gel solutions with photopolymerization catalysts (see recipe)
  • recipe10× electrophoresis buffer (see recipe)
  • Protein samples
  • recipeAcidicor recipebasic protein sample buffer (see recipes)
  • recipeCoomassie blue R‐250 stain solution (see recipe)
  • Gel rinse solution: 50% (v/v) methanol/7.5% (v/v) acetic acid
  • Gel destain solution: 5% (v/v) methanol/7.5% (v/v) acetic acid
  • Gel electrophoresis tank and glass plates (e.g., Bio‐Rad)
  • Spacers for casting gels (Aquebogue)
  • Gel casting box (Aquebogue)
  • Three‐channel peristaltic pump (e.g., ISCO Tris pump)
  • Light source to initiate photopolymerization of gels (e.g., two 15‐W blue fluorescent lamps)
  • Air‐displacement pipettor
  • Gel‐loading tip
  • Plastic boxes
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Literature Cited

   Brandts, J.F., Halvorson, H.R., and Brennan, M. 1975. Consideration of the possibility that the slow step in protein denaturation reactions is due to cis‐trans isomerism of proline residues. Biochemistry 14:4953‐4963.
   Creighton, T.E. 1979. Electrophoretic analysis of the unfolding of proteins by urea. J. Mol. Biol. 129:235‐264.
   Creighton, T.E. 1980. Kinetic study of protein unfolding and refolding using urea‐gradient electrophoresis. J. Mol. Biol. 137:61‐80.
   Creighton, T.E. 1992. Protein Folding. W.H. Freeman, New York.
   Creighton, T.E. and Shortle, D. 1994. Electrophoretic characterization of the denatured states of staphylococcal nuclease. J. Mol. Biol. 242:670‐682.
   Goldenberg, D.P. 1989. Analysis of protein conformation by gel electrophoresis. In Protein Structure: A Practical Approach (T.E. Creighton, ed.) pp. 225‐250. IRL Press, Oxford.
   Goldenberg, D.P. and Creighton, T.E. 1984. Gel electrophoresis in studies of protein conformation and folding. Anal. Biochem. 138:1‐18.
   Hollecker, M. and Creighton, T.E. 1982. Effect on protein stability of reversing the charge on amino groups. Biochim. Biophys. Acta 701:395‐404.
   Kim, P.S. and Baldwin, R.L. 1990. Intermediates in the folding reactions of small proteins. Annu. Rev. Biochem. 59:631‐660.
   Klemm, J.D., Wozniak, J.A., Alber, T., and Goldenberg, D.P. 1991. Correlation between mutational destabilization of phage T4 lysozyme and increased unfolding rates. Biochemistry 30:589‐594.
   Lyubimova, T., Caglio, S., Gelfi, C., Righetti, P.G., and Rabilloud, T. 1993. Photopolymerization of polyacrylamide gels with methylene blue. Electrophoresis 14:40‐50.
   Matthews, C.R. 1993. Pathways of protein folding. Annu. Rev. Biochem. 62:653‐683.
   McLellan, T. 1982. Electrophoresis buffers for polyacrylamide gels at various pH. Anal. Biochem. 126:94‐99.
   Pace, C.N., Shirley, B.A., and Thomson, J.A. 1989. Measuring the conformational stability of a protein. In Protein Structure: A Practical Approach. (T. E. Creighton, ed.) pp. 311‐330. IRL Press, Oxford.
   Privalov, P.L. 1989. Thermodynamic problems of protein structure. Annu. Rev. Biophys. Chem. 18:47‐69.
  Schellman, J.A. 1978. Solvent denaturation. Biopolymers 17:1305‐1322.
   Schellman, J.A. 1987. The thermodynamic stability of proteins. Annu. Rev. Biophys. Chem. 16:115‐137.
   Tanford, C. 1968. Protein denaturation. Adv. Protein Chem. 23:121‐282.
Key References
   Creighton, 1979. See above.
  The original description of urea‐gradient gels; includes extensive discussion of most of the important parameters and considerations and detailed analysis of the relationships between transition rates and electrophoretic patterns.
   Goldenberg and Creighton, 1984. See above.
  General review of gel electrophoresis and applications to studies of protein folding, including urea gradients.
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