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

  • Protocol
Basic Protein and Peptide Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 32))

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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. 2–4.

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References

  1. O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021.

    PubMed  Google Scholar 

  2. Celis, J. E. and Bravo, R., eds. (1984) Two-Dimensional Gel Electrophoresis of Proteins. Academic, New York.

    Google Scholar 

  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. Dunbar, B. S. (1987) Two-Dimensional Electrophoresis and Immunological Techniques. Plenum, New York.

    Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  7. Iborra, G. and Buhler, J. M. (1976) Protein subunit mapping. A sensitive high resolution method. Anal. Biochem. 74, 503–511.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

  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.

    Article  PubMed  Google Scholar 

  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. Görg, A., Postel, W., and Gunther, S. (1988) The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 9, 531–546.

    Article  PubMed  Google Scholar 

  13. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Cardiothoracic Surgery, National Heart and Lung Institute, Heart Science Centre, Harefield, UK

    Michael J. Dunn

Authors
  1. Michael J. Dunn
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Editor information

Editors and Affiliations

  1. University of Hertfordshire, Hatfield, UK

    John M. Walker

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© 1994 Humana Press Inc., Totowa, NJ

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Dunn, M.J. (1994). Two-Dimensional Polyacrylamide Gel Electrophoresis Using Flat-Bed Isoelectric Focusing in the First Dimension. In: Walker, J.M. (eds) Basic Protein and Peptide Protocols. Methods in Molecular Biology™, vol 32. Humana Press. https://doi.org/10.1385/0-89603-268-X:97

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  • DOI: https://doi.org/10.1385/0-89603-268-X:97

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-268-2

  • Online ISBN: 978-1-59259-519-8

  • eBook Packages: Springer Protocols

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