New Protein Techniques pp 203-216 | Cite as
Two-Dimensional Electrophoresis Using Immobilized pH Gradients in the First Dimension
- 1.3k Downloads
Abstract
The high-resolution capacity of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) based on the method of O’Farrell (1) makes it the method of choice for the analysis of complex protein mixtures (2, 3, 4). In the standard procedure, cylindrical tube isoelectric focusing (IEF) gels are used. Unfortunately this system suffers from severe cathodic drift resulting in pH gradients that do not extend above pH 8, resulting in concomitant loss of basic proteins from 2-D maps. Gradients can be extended to pH 10 by special treatment of the glass IEF tubes (5) or by using a horizontal flat-bed IEF (5, 6, 7). In spite of the improvement in resolution of basic proteins that can be achieved with these procedures, difficulties are still associated with the first-dimension IEF gels as a consequence of the characteristic properties of the synthetic carrier ampholytes used to generate the pH gradients. These include problems of batch reproducibility of ampholytes, irreproducibility of separations, difficulty in control of pH gradient stability and shape (i.e., “pH gradient engineering”), and the possibility of artifacts caused by protein-ampholyte interactions.
Keywords
Plastic Plate Complex Protein Mixture Plate Assembly Batch Reproducibility Silicon Rubber GasketReferences
- 1.O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021.PubMedGoogle Scholar
- 2.Dunn, M. J. and Burghes, A. H. M. (1983) High resolution two-dimensional polyacrylamide gel electrophoresis. I. Methodological procedures. Electrophoresis 4, 97–116.CrossRefGoogle Scholar
- 3.Dunn, M. J. and Burghes, A. H.M. (1983) High resolution two-dimensional polyacrylamide gel electrophoresis. II. Analysis and applications. Electrophoresis 4, 173–189.CrossRefGoogle Scholar
- 4.Dunn, M. J. and Burghes, A. H. M. (1986) High Resolution Two-Dimensional Polyacrylamide Gel Electrophoresis, in Gel Electrophoresis of Proteins (Dunn, M. J., ed.). Wright, Bristol.Google Scholar
- 5.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
- 6.Dunn, M. J., Burghes, A. H. M., Witkowski, J. A., and Dubowitz, V. (1985) Analysis of genetic muscle disorders by two-dimensional electrophoresis. Protides Biol. Fluids 32, 973–976.Google Scholar
- 7.Dunn, M. J. and Patel, K. (1987) Two-Dimensional Polyacrylamide Gel Electrophoresis Using Flatbed Isoelectric Focusing in the First Dimension, in Methods in Molecular Biology, vol. 3 (Walker, J. M., ed.). Humana, Clifton, New Jersey.Google Scholar
- 8.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
- 9.Righetti, P. G., Gelfi, C., and Gianazza, E. (1986) Conventional Isoelectric Focusing and Immobilized pH Gradients, in Gel Electrophoresis of Proteins (Dunn, M. J., ed.). Wright, Bristol.Google Scholar
- 10.Gianazza, E., Astrua-Testori, S., and Righetti, P. G. (1985) some more formulations for immobilized pH gradients. Electrophoresis 6, 103–146.CrossRefGoogle Scholar
- 11.Altland, K. and Rossmann, U. (1985) Hybrid isoelectric focusing in rehydrated immobilized pH gradients with added carrier ampholytes: Demonstration of human globins. Electrophoresis 6, 314–325.CrossRefGoogle Scholar
- 12.Fawcett, J. S. and Chrambach, A. (1985) Electrofocusing in wide pH range Immobiline gels containing carrier ampholytes. Protides Biol Fluids 33,439–4442.Google Scholar
- 13.Fawcett, J. S. and Chrambach, A. (1986) The voltage across wide pH range immobilized pH gradient gels and its modualtion through the addition of carrier ampholytes. Electrophoresis 7, 266–272.CrossRefGoogle Scholar
- 14.Dunn, M. J., Burghes, A. H. M., and Patel, K. (1985) Two-dimensional electrophoretic analysis of genetic muscle disorders. Protides Biol. Fluid 33, 479 82.Google Scholar
- 15.Dunn, M. J. and Patel, K. (1986) Two dimensional polyacrylamide gel electrophoresis using immobilized pH gradients in the first dimension. Protides Biol. Fluids 34, 695–699.Google Scholar
- 16.Gorg, A., Postel, W., and Weser, J. (1985) IEF and 2D-electro-phoresis with narrow and ultranarrow immobilized pH gradients for the analysis of genetic variants. Proteides Biol. Fluids 33, 467–470.Google Scholar
- 17.LKB Application Note Number 324 (1984) LKB Produkter AB, Bromma, Sweden.Google Scholar
- 18.Fawcett, J. S. and Chrambach, A. (1986) Simplified procedure for the prepation of immobilized pH gradient gels. Electrophoresis 7, 260–266.CrossRefGoogle Scholar
- 19.Altland, K. and Altland, A. (1984) Forming reproducible density and solute gradients by computer-controlled cooperation of stepmotor-driven burettes. Electrophoresis 5, 143–147.CrossRefGoogle Scholar
- 20.Altland, K. and Altland, A. (1984) Pouring reproducible gradients in gels under computer control: New devices for simultaneous delivery of two independent gradients, for more flexible slope and pH range of immobilized pH gradients. Clin. Chem. 30 2098–2103.PubMedGoogle Scholar
- 21.Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.PubMedCrossRefGoogle Scholar
- 22.Dunn, M. J., Patel, K., and Burghes, A. H. M. (1986) New Developments for the First Dimension of 2D-PAGE, in Progres Recents en Electrophorese Bidimensionelle (Galteau, M. M. and Siest, G. eds.) Presses Universitaires, Nancy.Google Scholar
- 23.Gelfi, C. and Righetti, P. G. (1984) Swelling kinetics of Immobiline gels for isoelectric focusing. Electrophoresis 5, 257–262.CrossRefGoogle Scholar