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Analysis of Membrane Proteins by Two-Dimensional Gels

  • Michael Fountoulakis
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Separation of a protein mixture by two-dimensional (2-D) gel electrophoresis is usually the first step of a proteomic analysis. The second step is the protein identification by mass spectrometry techniques, mainly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Two-dimensional electrophoresis comprises two steps (dimensions): (1) separation of the proteins on the basis of differences in their net charge, called isoelectric focusing (IEF), which is usually performed on immobilized pH gradient (IPG) strips; and (2) separation of the focused proteins on the basis of differences in their molecular masses, which is performed in sodium dodecyl sulfate (SDS) polyacrylamide gels. The major advantage of 2-D electrophoresis is that it enables the simultaneous separation and visualization of thousands of unknown protein forms. No other method can do that at the present time. On the other hand, protein detection in 2-D gels is limited because (1) the major components of a protein mixture are usually visualized and (2) the detection of the low- and high-molecular-mass proteins, as well as of the basic and hydrophobic proteins, is inefficient.

Keywords

Sodium Dodecyl Sulfate Acrylamide Solution Lithium Dodecyl Sulfate Zwitterionic Detergent Lithium Dodecyl Sulfate 
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.
    Herbert, B. (1999) Advances in protein solubilisation for two-dimensional electrophoresis. Electrophoresis 20, 660–663.PubMedCrossRefGoogle Scholar
  2. 2.
    Molloy, M. P. (2000) Two-dimensional electrophoresis of membrane proteins using immobilized pH gradients. Anal. Biochem. 280, 1–10.PubMedCrossRefGoogle Scholar
  3. 3.
    Santoni, V., Molloy, M., and Rabilloud, T. (2000) Membrane proteins and proteomics: un amour impossible? Electrophoresis 21, 1054–1070.PubMedCrossRefGoogle Scholar
  4. 4.
    Fountoulakis, M., and Gasser, R. (2003) Proteomic analysis of the cell envelope fraction of Escherichia coli. Amino Acids 24, 19–41.PubMedGoogle Scholar
  5. 5.
    Fountoulakis, M. (2000) Two-dimensional electrophoresis. In Encyclopedia of Separation Science, II/Electrophoresis, Academic Press, London, pp. 1356–1363.Google Scholar
  6. 6.
    Fountoulakis, M. and Takács, B. (2001) Effect of strong detergents and chaotropes on the protein detection in two-dimensional gels. Electrophoresis 22, 1593–1602.PubMedCrossRefGoogle Scholar
  7. 7.
    Chevallet, M., Santoni, V., Poinas, A., et al. (1998) New zwitterionic detergents improve the analysis of membrane proteins by two-dimensional electrophoresis. Electrophoresis 19, 1901–1909.PubMedCrossRefGoogle Scholar
  8. 8.
    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
  9. 9.
    Rabilloud, T., Blisnick, T., Heller, M., et al. (1999) Analysis of membrane proteins by two-dimensional electrophoresis: comparison of the proteins extracted from normal or Plasmodium falciparum-infected erythrocyte ghosts. Electrophoresis 20, 3603–3610.PubMedCrossRefGoogle Scholar
  10. 10.
    Ferro, M., Seigneurin-Berny, D., Rolland, N., et al. (2000) Organic solvent extraction as a versatile procedure to identify hydrophobic chloroplast membrane proteins. Electrophoresis 21, 3517–3526.PubMedCrossRefGoogle Scholar
  11. 11.
    Carboni, L., Piubelli, C., Righetti, P. G., Jansson, B., and Domenici, E. (2002) Proteomic analysis of rat brain tissue: comparison of protocols for two-dimensional gel electrophoresis analysis based on different solubilizing agents. Electrophoresis 23, 4132–4141.PubMedCrossRefGoogle Scholar
  12. 12.
    Hartinger, J., Stenius, K., Högemann, D., and Jahn, R. (1996) 16-BAC/SDS-PAGE: a twodimensional gel electrophoresis system suitable for the separation of integral membrane proteins. Anal. Biochem. 240, 126–133.PubMedCrossRefGoogle Scholar
  13. 13.
    Fountoulakis, M. (2001) Proteomics: Current technologies and applications in neurological disorders and toxicology. Amino Acids 21, 363–381.PubMedCrossRefGoogle Scholar
  14. 14.
    Fountoulakis, M. and Takcs, B. (2002) Enrichment and proteomic analysis of low-abundance bacterial proteins. Methods Enzymol. 358, 288–306.PubMedCrossRefGoogle Scholar
  15. 15.
    Fountoulakis M. (2004) Application of proteomics technologies in the investigation of the brain. Mass Spectrom. Rev. 23(4), 231–258.PubMedCrossRefGoogle Scholar
  16. 16.
    Langen, H., Roeder, D., Juranville, J.-F., and Fountoulakis, M. (1997). Effect of the protein application mode and the acrylamide concentration on the resolution of protein spots separated by two-dimensional gel electrophoresis. Electrophoresis 18, 2085–2090.PubMedCrossRefGoogle Scholar
  17. 17.
    Westermeier R. (ed). (1993) Electrophoresis in Practice VCH Verlagsgesellschaft, Weinheim, pp. 215–263.Google Scholar
  18. 18.
    Schaegger, H. and von Jagow, G. (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368–379.CrossRefGoogle Scholar
  19. 19.
    Kyte, J. and Doolittle, R. F. (1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105–132.PubMedCrossRefGoogle Scholar
  20. 20.
    Jiang, L., He, L., and Fountoulakis, M. (2004) Comparison of protein precipitation methods for sample preparation prior to proteomic analysis. J. Chromatogr. A 1023, 317–320.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2005

Authors and Affiliations

  • Michael Fountoulakis
    • 1
    • 2
  1. 1.F. Hoffman-LaRoche Ltd., Center for Medical GenomicsBaselSwitzerland
  2. 2.Foundation for Biomedical Research of the Academy of AthensGreece

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