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Preparation of Bacterial Samples for 2-D PAGE

  • Brian Berg Vandahl
  • Gunna Christiansen
  • Svend Birkelund
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Sample preparation is a very crucial step in two-dimensional (2-D) gel electrophoresis, in which the proteins of the sample must be brought into a state where they can be separated by isoelectric focusing in the first dimension. That is, they must be denatured, reduced, and solubilized, and they must be kept so during electrophoresis without changing their pI. The sample buffer for this purpose is traditionally called the lysis buffer.

Keywords

Sodium Dodecyl Sulfate Lysis Buffer Tris Base Urea Solution Bromphenol Blue 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Righetti, P. G., Castagna A., Herbert B., Reymond F., and Rossier J. S. (2003) Prefractionation techniques in proteome analysis. Proteomics 3, 1397–1407.PubMedCrossRefGoogle Scholar
  2. 2.
    Castellanos-Serra, L., and Paz-Lago, D. (2002) Inhibition of unwanted proteolysis during sample preparation: evaluation of its efficiency in challenge experiments. Electrophoresis 23, 1745–1753.PubMedCrossRefGoogle Scholar
  3. 3.
    Harder, A., Wildgruber, R., Nawrocki, A., Fey, S. J., Larsen, P. M., and Gorg, A. (1999) Comparison of yeast cell protein solubilization procedures for two-dimensional electrophoresis. Electrophoresis 20, 826–829.PubMedCrossRefGoogle Scholar
  4. 4.
    O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol Chem. 250,4007–4021.PubMedGoogle Scholar
  5. 5.
    Ames, G. F. and Nikaido, K. (1976) Two-dimensional gel electrophoresis of membrane proteins. Biochemistry 15, 616–623.PubMedCrossRefGoogle Scholar
  6. 6.
    Jacobs, D. I., van Rijssen, M. S., van der Heijden, R., and Verpoorte, R. (2001) Sequential solubilization of proteins precipitated with trichloroacetic acid in acetone from cultured Catharanthus roseus cells yields 52% more spots after two-dimensional electrophoresis. Proteomics 1, 1345–1350.PubMedCrossRefGoogle Scholar
  7. 7.
    McCarthy, J., Hopwood, F., Oxley, D., et al. (2003) Carbamylation of proteins in 2-D electrophoresis-myth or reality? J. Proteome Res. 2, 239–242.PubMedCrossRefGoogle Scholar
  8. 8.
    Rabilloud, T., Adessi, C., Giraudel, A., and Lunardi, J. (1997) Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 18, 307–316.PubMedCrossRefGoogle Scholar
  9. 9.
    Herbert, B. R., Molloy, M. P., Gooley, A. A., Walsh, B. J., Bryson, W. G., and Williams, K. L. (1998) Improved protein solubility in two-dimensional electrophoresis using tributyl phosphine as reducing agent. Electrophoresis 19, 845–851.PubMedCrossRefGoogle Scholar
  10. 10.
    Tastet, C., Charmont, S., Chevallet, M., Luche, S., and Rabilloud, T. (2003) Structureefficiency relationships of zwitterionic detergents as protein solubilizers in two-dimensional electrophoresis. Proteomics 3, 111–121.PubMedCrossRefGoogle Scholar
  11. 11.
    Luche, S., Santoni, V., and Rabilloud, T. (2003) Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in two-dimensional electrophoresis. Proteomics 3, 249–253.PubMedCrossRefGoogle Scholar
  12. 12.
    Vandahl, B. B., Birkelund, S., and Christiansen, G. (2002) Proteome analysis of Chlamydia pneumoniae. Methods Enzymol. 358, 277–288.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2005

Authors and Affiliations

  • Brian Berg Vandahl
    • 1
  • Gunna Christiansen
    • 2
  • Svend Birkelund
    • 1
  1. 1.Department of Medical Microbiology and ImmunologyUniversity of Aarhus, DenmarkLoke Diagnostics ApSDenmark
  2. 2.Department of Medical Microbiology and ImmunologyUniversity of AarhusDenmark

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