Advertisement

Large-Format 2-D Polyacrylamide Gel Electrophoresis

  • Henry Brzeski
  • Stephen Russell
  • Anthony G. Sullivan
  • Richard I. Somiari
  • Craig D. Shriver
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Proteins are composed of different numbers of neutral positively and negatively charged amino acids. Therefore, proteins vary widely in size and have either positive, negative, or zero net charge, depending on the pH of their surroundings. The original two-dimensional gel electrophoresis format was developed almost 30 years ago (1) to exploit this variation in protein charge and size for separation purposes. The isoelectric point of a protein (pI) is the pH at which it has a net zero charge, which, for the majority of proteins, lies between pH 4.0 and 8.0. The sizes of proteins vary widely (10–500 kDa), with an average molecular weight of approx 50 kDa. These two mutually independent properties are exploited by firstly denaturing proteins in urea and then subjecting them to an electric field in a pH gradient established in a low-concentration polyacrylamide gel (originally this pH gradient was formed, using ampholytes, in situ, but now precast immobilized pH gradient [IPG] strips [2, 3, 4] are found to be more reliable). In this case, all but the very largest proteins can migrate freely until they reach a pH at which they have no net charge (isoelectric focusing [IEF]). After completion of the focusing, the proteins are denatured in situ, their native charge is saturated with the anionic detergent sodium dodecyl sulphate (SDS), and then the gel is layered, perpendicular to the direction of focusing, on a higher-concentration polyacrylamide gel, and the focused proteins are separated on the basis of size. This gives rise to discrete spots representing one (or perhaps a very small number) of different proteins.

Keywords

Sodium Dodecyl Sulphate Anionic Detergent Sodium Dodecyl Sulphate Acrylamide Solution Strip Holder Acrylamide Polymerization 
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.
    O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021.Google Scholar
  2. 2.
    Westermeier, R., Ek, K., Righetti, P. G., Gianazza, E., Görg, A., and Postel, W. (1982) Isoelectric focusing in immobilized pH gradients: principle, methodology and some applications. J. Biochem. Biophys. 6(317), 339.Google Scholar
  3. 3.
    Görg, A., Postel, W., Gnther, S., and Weser, J. (1985) Improved horizontal two-dimensional electrophoresis with hybrid isoelectric focusing in immobilized pH gradients in the first dimension and laying-on transfer to the second dimension. Electrophoresis 6, 599–604.CrossRefGoogle Scholar
  4. 4.
    Görg, A., Postel, W., and Gnther, S. (1988) The current state of two-dimensional immobilized pH gradients. Electrophoresis 9, 531–546.PubMedCrossRefGoogle Scholar
  5. 5.
    Lilley, K. S., Razzaq, A., and Dupree, P. (2002) Two-dimensional gel electrophoresis: recent advances in sample preparation, detection and quantitation. Curr. Opin. Chem. Biol. 6(1), 46–50.PubMedCrossRefGoogle Scholar
  6. 6.
    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), 1375–1378.PubMedCrossRefGoogle Scholar
  7. 7.
    Sanchez, J.-C., Rouge, V., Pisteur, M., et al. (1997) Improved and simplified in-gel sample application using reswelling of dry immobilized pH gradients. Electrophoresis 18, 324–327.PubMedCrossRefGoogle Scholar
  8. 8.
    Rabilloud, T., Valette, C., and Lawrence, J. J. (1994) Sample application by in-gel rehydration improves the resolution of two-dimensional electrophoresis with immobilized pH gradients in the first dimension. Electrophoresis 15, 1552–1558.PubMedCrossRefGoogle Scholar
  9. 9.
    Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.PubMedCrossRefGoogle Scholar
  10. 10.
    Molloy, M. P., Herbert, B. R., Walsh, B. J., et al. (1998) Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis. Electrophoresis 19, 837–844.PubMedCrossRefGoogle Scholar
  11. 11.
    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
  12. 12.
    Rabilloud, T. (1998) Use of thiourea to increase the solubility of membrane proteins in two-dimensional electrophoresis. Electrophoresis 19, 759–760.Google Scholar
  13. 13.
    Görg, A., Boguth, G., Obermaier, C., Harder, A., and Weiss, W. (1998) 2-D electrophoresis with immobilized pH gradients using IPGphor isoelectric focusing system. Life Science News 1, 4–6.Google Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2005

Authors and Affiliations

  • Henry Brzeski
    • 3
  • Stephen Russell
    • 3
  • Anthony G. Sullivan
    • 2
    • 1
  • Richard I. Somiari
    • 4
  • Craig D. Shriver
    • 5
  1. 1.Functional Genomics and Proteomics Unit, Windber Research InstituteWindber
  2. 2.Functional Genomics and Proteomics Unit, Thermoelectron Training InstituteWest Palm Beach
  3. 3.Functional Genomics and Proteomics Unit, Windber ResearchInstituteWindber
  4. 4.Functional Genomics and Protemics Unit, ITSI-BiosciencesJohnstown
  5. 5.Clinical Breast Care Project, Walter Reed Army Medical CenterWashington,DC

Personalised recommendations