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Two-Dimensional Polyacrylamide Gel Electrophoresis Using Flat-Bed Isoelectric Focusing in the First Dimension

  • Michael J. Dunn
Part of the Methods in Molecular Biology™ book series (MIMB, volume 32)

Abstract

Electrophoretic techniques are, perhaps, the most important group of procedures for the separation and analysis of complex protein mixtures because of their high resolution capacity. This resolving power is maximized in high-resolution, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) based on the method developed by O’Farrell (1). This methodology involves a combination of a first-dimension separation in cylindrical isoelectric focusing (IEF) gels containing 9M urea and 2% (w/v) of the detergent NP-40 with discontinuous polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE) in the second dimension. This technique is described in  Chapter 11 and is reviewed in refs. 2, 3, 4.

Keywords

Cover Sheet Cooling Plate Lane Marking Silicon Fluid High Resolution Capacity 
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.PubMedGoogle Scholar
  2. 2.
    Celis, J. E. and Bravo, R., eds. (1984) Two-Dimensional Gel Electrophoresis of Proteins. Academic, New York.Google Scholar
  3. 3.
    Dunn, M. J. (1987) Two-dimensional polyacrylamide gel electrophoresis, in Advances in Electrophoresis, vol. 1 (Chrambach, A., Dunn, M. J., and Radola, B. J., eds.), VCH Verlagsgesellschaft, Weinheim, Germany, pp. 1–109.Google Scholar
  4. 4.
    Dunbar, B. S. (1987) Two-Dimensional Electrophoresis and Immunological Techniques. Plenum, New York.Google Scholar
  5. 5.
    O’Farrell, P., Goodman, M. M., and O’Farrell, P. H. (1977) High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12, 1133–1142.PubMedCrossRefGoogle Scholar
  6. 6.
    Goldsmith, M. R., Rattner, E. C., Macy, M., Koehler, D., Balikov, S. R., and Bock, S. C. (1979) Two-dimensional electrophoresis of small molecular weight proteins. Anal. Biochem. 99, 33–40.PubMedCrossRefGoogle Scholar
  7. 7.
    Iborra, G. and Buhler, J. M. (1976) Protein subunit mapping. A sensitive high resolution method. Anal. Biochem. 74, 503–511.PubMedCrossRefGoogle Scholar
  8. 8.
    Rangel-Aldao, R., Kupiec, J. W., and Rosen, O. M. (1979) Resolution of the phosphorylated and dephosphorylated cAMP binding proteins of bovine cardiac muscle by affinity labelling and two-dimensional electrophoresis. J. Biol. Chem. 254, 2499–2508.PubMedGoogle Scholar
  9. 9.
    Burghes, A. H. M., Dunn, M. J., and Dubowitz, V. (1982) Enhancement of resolution in two-dimensional gel electrophoresis and simultaneous resolution of acidic and basic proteins. Electrophoresis 3, 354–363.CrossRefGoogle Scholar
  10. 10.
    Görg, A., Postel, W., Westermeier, R., Gianazza, E., and Righetti, P. G. (1980) Gel gradient electrophoresis, isoelectric focusing and two-dimensional techniques in horizontal, ultrathin polyacrylamide layers. J. Biochem. Biophys. Methods 3, 273–284.PubMedCrossRefGoogle Scholar
  11. 11.
    Görg, A., Postel, W., and Westermeier, R. (1981) SDS-gel gradient electrophoresis, isoelectric focusing and high-resolution two-dimensional electrophoresis in horizontal ultrathin-layer polyacrylamide gels, in Electrophoresis ′81 (Allen, R. C. and Arnaud, P., ed.), DeGruyter, Berlin, pp. 259–270.Google Scholar
  12. 12.
    Görg, A., Postel, W., and Gunther, S. (1988) The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 9, 531–546.PubMedCrossRefGoogle Scholar
  13. 13.
    Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.PubMedCrossRefGoogle Scholar
  14. 14.
    Burghes, A. H. M., Patel, K., and Dunn, M. J. (1985) Comparison of focusing in buffers and synthetic carrier ampholytes for use in the first dimension of two-dimensional polyacrylamide gel electrophoresis. Electrophoresis 6, 453–461.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1994

Authors and Affiliations

  • Michael J. Dunn
    • 1
  1. 1.Department of Cardiothoracic Surgery, National Heart and Lung InstituteHeart Science CentreHarefieldUK

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