Abstract
Two-dimensional electrophoresis (2-DE) of proteins is used for several purposes, such as resolving a distinct group of proteins (e.g., serum proteins), revealing the heterogeneity of a particular protein (e.g., actin, transferrin), purifying a protein, or testing the purity of a protein gained by other methods. However, the most exceptional feature of this method is its potentiality to resolve all the various proteins of a certain cell type or tissue. It was this particular feature of the 2-DE technique developed in 1975 (1,2) that opened the way to study many of the biological problems of present interest under a new aspect: Problems of gene expression, gene regulation, genetic variation, cell differentiation, embryonic development, pathogenesis of certain diseases, and other fields could be studied on the basis of a broad spectrum and a representative number of proteins, whereas previously studies like these were performed on selected proteins, often selected because of their easy accessibility and considered as model proteins (e.g. hemoglobin). Moreover, when simple procedures for extracting cell or tissue proteins were used, i.e., procedures that avoid steps, such as protein precipitation, lyophilization, dialyses, or chromatographic fractionations, 2-DE protein patterns revealed the individual proteins in quantities that reflect, at least to some extent, the relative concentration of these proteins in the cells or tissues. Quantitative data on gene expression offer an important parameter for all studies in cell biology.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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, 211–243.
O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021.
Fields, C, Adams, M. D., White, O., and Venter, J. C. (1994) How many genes in the human genome? Nature Genetics 7, 345–346.
Anderson, W. F. (1994) Genes, genes, and more genes (Editorial). Hum. Gene Ther. 5, 1077–1078.
McConkey, E. H. (1979) Double-label autoradiography for comparison of complex protein mixtures after gel electrophoresis. Anal. Biochem. 96, 39–44.
Hirsh, A. G. (1984) On the possibility of the production of many rare proteins by higher eukaryotes. J. Theor. Biol. 110, 399–410.
Jungblut, P. R., Prehm, J., and Klose, J. (1987) An attempt to resolve all the various proteins of a single human cell type by two-dimensional electrophoresis. Biol. Chem. Hoppe-Seyler 368, 439.
Duncan, R. and McConkey, H. (1982) How many proteins are there in a typical mammalian cell? Clin. Chem. 28, 749–755.
Klose, J. (1983) High resolution of complex protein solutions by two-dimensional electrophoresis, in Modern Methods in Protein Chemistry (Tschesche, H., ed.), Review Articles, vol. 1, Walter de Gryuter Verlag, Berlin, pp. 49–78.
Klose, J. and Kobalz, U. (1995) Two-dimensional electrophoresis of proteins: An updated protocol and implications for a functional analysis of the genome. Electrophoresis 16, 1034–1059.
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, 307–317.
Young, D. A., Voris, B. P., Maytin, E. V., and Colbert, R. A. (1983) Very-high-resolution two-dimensional electrophoretic separation of proteins on giant cells. Methods Enzymol. 91, 190–214.
Klose, J. and Feller, M. (1981) Two-dimensional electrophoresis of membrane and cytosol proteins of mouse liver and brain. Electrophoresis 2, 12–24.
Rabilloud, T. (1994) Two-dimensional electrophoresis of basic proteins with equilibrium isoelectric focusing in carrier ampholyte-pH gradients. Electrophoresis 15, 278–282.
Laemmli, V. (1970) Cleavage of the structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.
Heukeshoven, J. and Dernick, R. (1985) Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 6, 103–112.
Jungblut, P. and Seifert, R. (1990) Analysis by high-resolution two-dimensional electrophoresis of differentiation-dependent alterations in cytosolic protein pattern of HL-60 leukemic cells. J. Biochem. Biophys. Methods 21, 47–58.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1999 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Klose, J. (1999). Large-Gel 2-D Electrophoresis. In: Link, A.J. (eds) 2-D Proteome Analysis Protocols. Methods in Molecular Biology, vol 112. Humana Press. https://doi.org/10.1385/1-59259-584-7:147
Download citation
DOI: https://doi.org/10.1385/1-59259-584-7:147
Publisher Name: Humana Press
Print ISBN: 978-0-89603-524-9
Online ISBN: 978-1-59259-584-6
eBook Packages: Springer Protocols