Two-Dimensional Polyacrylamide Gel Electrophoresis for the Separation of Proteins for Chemical Characterization

  • Michael J. Dunn
Part of the Methods in Molecular Biology™ book series (MIMB, volume 211)

Abstract

The first complete genome, that of the bacterium Hemophilus influenzae, was published in 1995 (1). We now have the complete genomic sequences for more than 80 prokaryotic and eukaryotic organisms, and a major milestone has been reached recently with the completion of the human genome (2,3). A major challenge in the post-genome era will be to elucidate the biological function of the large number of novel gene products that have been revealed by the genome sequencing initiatives, to understand their role in health and disease, and to exploit this information to develop new therapeutic agents. The assignment of protein function will require detailed and direct analysis of the patterns of expression, interaction, localization, and structure of the proteins encoded by genomes; the area now known as & proteomics& (4).

Keywords

Crystallization Urea Glutathione Cysteine Electrophoresis 

References

  1. 1.
    Fleischmann, R. D., et al. (1995) Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269, 496–512PubMedCrossRefGoogle Scholar
  2. 2.
    Venter, J. C. et al. (2001) The sequence of the human genome. Science 291, 1304–1351.PubMedCrossRefGoogle Scholar
  3. 3.
    International Human Genome Sequencing Consortium (2001) Initial sequencing and analysis of the human genome. Nature 409, 860–922.CrossRefGoogle Scholar
  4. 4.
    Banks, R., Dunn, M. J., Hochstrasser, D. F., et al. (2000) Proteomics: new perspectives, new biomedical opportunities. Lancet 356, 1749–1756.PubMedCrossRefGoogle Scholar
  5. 5.
    Merchant, M. and Weinberger, S. R. (2000) Recent advances in surface enhanced laser-desorption/ionization time-of-flight mass spectrometry. Electrophoresis 21, 1165–1177.CrossRefGoogle Scholar
  6. 6.
    Nelson, R. W., Nedelkov, D., and Tubbs, K. A. (2000) Biosensor chip mass spectrometry: a chip-based approach. Electrophoresis 21, 1155–1163.PubMedCrossRefGoogle Scholar
  7. 7.
    Link, A. J., Eng, J., Schieltz, D. M., et al. (1999) Direct analysis of protein complexes using mass spectrometry. Nature Biotechnol. 17, 676–682.CrossRefGoogle Scholar
  8. 8.
    Gygi, S. P., Rist, B., Gerber, S. A., et al. (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nature Biotechnol. 17, 994–999.CrossRefGoogle Scholar
  9. 9.
    Rigaut, G., Shevchenko, A., Rutz, B., et al. (1999) A generic protein purification method for protein complex characterization and proteome exploration. Nature Biotechnol. 17, 1030–1032.CrossRefGoogle Scholar
  10. 10.
    Uetz, P., Giot, L., Cagney, G., et al. (2000) A comprehensive analysis of proteinprotein interactions in Saccharomyces cerevisiae. Nature 403, 623–627.PubMedCrossRefGoogle Scholar
  11. 11.
    Dunn, M. J. and Görg, A. (2001) Two-dimensional polyacrylamide gel electrophoresis for proteome analysis, in Proteomics, From Protein Sequence to Function (Pennington, S. R. and Dunn, M. J., eds.), BIOS Scientific Publishers, Oxford, pp. 43–63.Google Scholar
  12. 12.
    Patton, W. F. (2001) Detecting proteins in polyacrylamide gels and on electroblot membranes, in Proteomics, From Protein Sequence to Function (Pennington, S. R. and Dunn, M. J., eds.), BIOS Scientific Publishers, Oxford, pp. 65–86.Google Scholar
  13. 13.
    Dunn, M. J. (1992) The analysis of two-dimensional polyacrylamide gels for the construction of protein databases, in Microcomputers in Biochemistry (Bryce, C. F. A., ed.), IRL Press, Oxford, pp. 215–242.Google Scholar
  14. 14.
    Wilkins, M. R. and Gooley, A. (1997) Protein identification in proteome analysis, in Proteome Research: New Frontiers in Functional Genomics (Wilkins, M. R., Williams, K. L., Appel, R. D. and Hochstrasser, D. F., eds.), Springer-Verlag, Berlin, pp. 35–64.Google Scholar
  15. 15.
    Patterson, S. D., Aebersold, R., and Goodlett, D. R. (2001) Mass spectrometrybased methods for protein identification and phosphorylation site analysis, in Proteomics, From Protein Sequence to Function (Pennington, S. R. and Dunn, M. J., eds.), BIOS Scientific Publishers, Oxford, pp. 87–130.Google Scholar
  16. 16.
    O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021.Google Scholar
  17. 17.
    Klose, J. (1975) Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals. Humangenetik 26, 231–243.PubMedGoogle Scholar
  18. 18.
    Görg, A., Obermaier, C., Boguth, G., et al. (2000) The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21, 1037–1053.PubMedCrossRefGoogle Scholar
  19. 19.
    Görg, A., Obermaier, C., Boguth, G., et a;. (1997) Very alkaline immobilized pH gradients for two-dimensional electrophoresis of ribosomal and nuclear proteins. Electrophoresis 18, 328–37.PubMedCrossRefGoogle Scholar
  20. 20.
    Hanash, S. M., Strahler, J. R., Neel, J. V., et al. (1991) Highly resolving two-dimensional gels for protein sequencing. Proc. Natl. Acad. Sci. USA 88, 5709–5713.PubMedCrossRefGoogle Scholar
  21. 21.
    Bjellqvist, B., Sanchez, J.-C., Pasquali, C., et al. (1993) Micropreparative two-dimensional electrophoresis allowing the separation of samples containing milligram amounts of proteins. Electrophoresis 14, 1375–1378.PubMedCrossRefGoogle Scholar
  22. 22.
    Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.PubMedCrossRefGoogle Scholar
  23. 23.
    Wildgruber, R., Harder, A., Obermaier, C., et al. (2000) Towards higher resolution: 2D-Electrophoresis of Saccharomyces cerevisiae proteins using overlapping narrow IPG’s. Electrophoresis 21, 2610–2616.PubMedCrossRefGoogle Scholar
  24. 24.
    Cordwell, S. J., Nouwens, A. S., Verrils, N. M., et al. (2000) Sub-proteomics based upon protein cellular location and relative solubilities in conjunction with composite two-dimensional gels. Electrophoresis 21, 1094–1103.PubMedCrossRefGoogle Scholar
  25. 25.
    Santoni, V., Molloy, M., and Rabilloud, T. (2000) Membrane proteins and proteomics: un amour impossible? Electrophoresis 21, 1054–1070.CrossRefGoogle Scholar
  26. 26.
    Görg, A., Postel, W., Friedrich, C., et al. (1991) Temperature-dependent spot positional variability in two-dimensional polypeptide gel patterns. Electrophoresis 12, 653–658.PubMedCrossRefGoogle Scholar
  27. 27.
    Patterson, S. D. (1994) From electrophoretically separated protein to identification: Strategies for sequence and mass analysis. Anal. Biochem. 221, 1–15.PubMedCrossRefGoogle Scholar
  28. 28.
    Patton, W. F. (2000) A thousand points of light: The application of fluorescence detection technologies to two-dimensional gel electrophoresis and proteomics. Electrophoresis 21, 1123–1144.PubMedCrossRefGoogle Scholar
  29. 29.
    Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal. Chem. 68, 85–858.CrossRefGoogle Scholar
  30. 30.
    Yan, J. X., Wait, R., Berkelman, T., et al. (2000) A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ ionization and electrospray ionization-mass spectrometry. Electrophoresis 21, 3666–3672.PubMedCrossRefGoogle Scholar
  31. 31.
    Yan, J. X., Harry, R. A., Spibey, C., and Dunn, M. J. (2000) Postelectrophoretic staining of proteins separated by two-dimensional gel electrophoresis using SYPRO dyes. Electrophoresis 21, 3657–3665.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2003

Authors and Affiliations

  • Michael J. Dunn
    • 1
  1. 1.Institute of PsychiatryKings College LondonLondonUK

Personalised recommendations