Summary
Our two-dimensional gel electrophoresis (2DE) protocol has been continuously improved in our laboratory since its inception 30 years ago. An updated version is presented here. This protocol is a result of our experience in proteome analysis of tissue extracts, cultured cells (mammalian, yeast, and bacteria), cellular organelles, and subcellular fractions. Many modifications and suggestions emerging in our lab as well as in the literature were tested and integrated into our improved protocol if helpful. Importantly we use (a) large (46 × 30 cm) gels to achieve a high resolution and (b) ready-made gel solutions produced in large batches and stored frozen, a prerequisite, among others, for our very high reproducibility. Employing the 2DE method described here we demonstrated that protein patterns separating more than 10,000 protein spots can be obtained from mouse tissue. This is the highest resolution reported in the literature for 2DE of complex protein mixtures so far. Our 2DE patterns are of high quality with regard to spot shape and intensity as well as background. The reproducibility of the protein patterns is shown to be extremely satisfactory. New staining methods such as differential in gel electrophoresis (DIGE) and the latest 2DE gel evaluation software are compatible to our 2DE protocol. Using suitable staining protocols proteins can easily be identified by mass spectrometry.
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, 231–43.
O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins, J Biol Chem 250, 4007–21.
Zabel, C. (2006) Proteins are specifically altered in disease – really? Expert Rev Proteomics 3, 469–70.
Zabel, C., Sagi, D., Kaindl, A. M., Steireif, N., Klare, Y., Mao, L., et al (2006) Comparative proteomics in neurodegenerative and non-neurodegenerative diseases suggest nodal point proteins in regulatory networking, J Proteome Res 5, 1948–58.
Zabel, C., Chamrad, D. C., Priller, J., Woodman, B., Meyer, H. E., Bates, G. P., et al (2002) Alterations in the mouse and human proteome caused by Huntington’s disease, Mol Cell Proteomics 1, 366–75.
Lander, E. S., Linton, L. M., Birren, B., Nusbaum, C., Zody, M. C., Baldwin, J., et al (2001) Initial sequencing and analysis of the human genome, Nature 409, 860–921.
Venter, J. C., Adams, M. D., Myers, E. W., Li, P. W., Mural, R. J., Sutton, G. G., et al (2001) The sequence of the human genome, Science 291, 1304–51.
Stein, L. D. (2004) Human genome: end of the beginning, Nature 431, 915–6.
Gibson, F., Anderson, L., Babnigg, G., Baker, M., Berth, M., Binz, P.A., et al. (2007) MIAPE: gel electrophoresis, Nat Biotechnol in Press
Anderson, W. F. (1994) Genes, genes, and more genes, Hum Gene Ther 5, 1077–8.
Klose, J., Nock, C., Herrmann, M., Stuhler, K., Marcus, K., Bluggel, M., et al (2002) Genetic analysis of the mouse brain proteome, Nat Genet 30, 385–93.
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, J Biol Chem Hoppe-Seyler 368, 439.
Duncan, R., and McConkey, E. H.(1982) How many proteins are there in a typical mammalian cell?Clin Chem 28, 749–55.
Sitek, B., Potthoff, S., Schulenborg, T., Stegbauer, J., Vinke, T., Rump, L. C., et al (2006) Novel approaches to analyse glomerular proteins from smallest scale murine and human samples using DIGE saturation labelling, Proteomics 6, 4337–45.
Sitek, B., Luttges, J., Marcus, K., Kloppel, G., Schmiegel, W., Meyer, H. E., et al (2005) Application of fluorescence difference gel electrophoresis saturation labelling for the analysis of microdissected precursor lesions of pancreatic ductal adenocarcinoma, Proteomics 5, 2665–79.
Klose, J. (1983) High resolution of complex protein solutions by two-dimensional electrophoresis. In Modern Methods in Protein Chemistry - Review Articles (Tschesche, H. ed.), walter de Gruyter Verlag, Berlin and New York, 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, 1034–59.
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, 307–16.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227, 680–5.
Heukeshoven, J., and Dernick, R. (1985) Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining, Electrophoresis 6, 103–12.
Jungblut, P. R., 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.
Nebrich, G., Herrmann, M., Sagi, D., Klose, J., and Giavalisco, P. (2007) High MS-compatibility of silver nitrate-stained protein spots from 2-DE gels using ZipPlates and AnchorChips for successful protein identification, Electrophoresis 28, 1607–14.
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, 278–82.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Zabel, C., Klose, J. (2009). High-Resolution Large-Gel 2DE. In: Tyther, R., Sheehan, D. (eds) Two-Dimensional Electrophoresis Protocols. Methods in Molecular Biology, vol 519. Humana Press. https://doi.org/10.1007/978-1-59745-281-6_20
Download citation
DOI: https://doi.org/10.1007/978-1-59745-281-6_20
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-58829-937-6
Online ISBN: 978-1-59745-281-6
eBook Packages: Springer Protocols