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2D DIGE Analysis of Protein Extracts from Muscle Tissue

  • Cecilia GelfiEmail author
  • Sara De Palma
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 854)

Abstract

2D DIGE, two-dimensional difference gel electrophoresis, is a technology used to study the protein expression on two-dimensional gels. Protein samples are labeled with different color fluorescent dyes designed not to affect the relative migration of proteins during electrophoresis. Here, we describe the practical procedures necessary to perform a 2D DIGE experiment for a muscle tissue protein extract followed by CyDye DIGE fluors minimal labeling and the analysis of 2D DIGE gels for the assessment of quantitative differences.

Key words

2D DIGE 2D electrophoresis CyDye DIGE fluors Differential analysis Muscle proteome 

Notes

Acknowledgements

This work was supported by the Telethon foundation (grant N. GGP08107D to C.G.), EU community (grant BIO-NMD N. 241665 to C.G.), and Italian Ministry of University and Scientific Research (grant FIRB RBRN07BMCT to C.G.).

References

  1. 1.
    Unlu, M., Morgan, M. E., and Minden, J. S. (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts, Electrophoresis 18, 2071–2077.PubMedCrossRefGoogle Scholar
  2. 2.
    Garrels, J. I. (1979) Two dimensional gel electrophoresis and computer analysis of proteins synthesized by clonal cell lines, J Biol Chem 254, 7961–7977.PubMedGoogle Scholar
  3. 3.
    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, 231–243.Google Scholar
  4. 4.
    O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins, J Biol Chem 250, 4007–4021.PubMedGoogle Scholar
  5. 5.
    Gharbi, S., Gaffney, P., Yang, A., Zvelebil, M. J., Cramer, R., Waterfield, M. D., and Timms, J. F. (2002) Evaluation of two-dimensional differential gel electrophoresis for proteomic expression analysis of a model breast cancer cell system, Mol Cell Proteomics 1, 91–98.PubMedCrossRefGoogle Scholar
  6. 6.
    Zhou, G., Li, H., DeCamp, D., Chen, S., Shu, H., Gong, Y., Flaig, M., Gillespie, J. W., Hu, N., Taylor, P. R., Emmert-Buck, M. R., Liotta, L. A., Petricoin, E. F., 3 rd, and Zhao, Y. (2002) 2D differential in-gel electrophoresis for the identification of esophageal scans cell ­cancer-specific protein markers, Mol Cell Proteomics 1, 117–124.PubMedCrossRefGoogle Scholar
  7. 7.
    Swatton, J. E., Prabakaran, S., Karp, N. A., Lilley, K. S., and Bahn, S. (2004) Protein profiling of human postmortem brain using 2-dimensional fluorescence difference gel ­electrophoresis (2-D DIGE), Mol Psychiatry 9, 128–143.PubMedCrossRefGoogle Scholar
  8. 8.
    Alban, A., David, S. O., Bjorkesten, L., Andersson, C., Sloge, E., Lewis, S., and Currie, I. (2003) A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard, Proteomics 3, 36–44.PubMedCrossRefGoogle Scholar
  9. 9.
    Knowles, M. R., Cervino, S., Skynner, H. A., Hunt, S. P., de Felipe, C., Salim, K., Meneses-Lorente, G., McAllister, G., and Guest, P. C. (2003) Multiplex proteomic analysis by two-dimensional differential in-gel electrophoresis, Proteomics 3, 1162–1171.PubMedCrossRefGoogle Scholar
  10. 10.
    Marouga, R., David, S., and Hawkins, E. (2005) The development of the DIGE system: 2D fluorescence difference gel analysis technology, Anal Bioanal Chem 382, 669–678.PubMedCrossRefGoogle Scholar
  11. 11.
    Yan, J. X., Devenish, A. T., Wait, R., Stone, T., Lewis, S., and Fowler, S. (2002) Fluorescence two-dimensional difference gel electrophoresis and mass spectrometry based proteomic ­analysis of Escherichia coli, Proteomics 2, 1682–1698.PubMedCrossRefGoogle Scholar
  12. 12.
    Friedman, D. B., Hill, S., Keller, J. W., Merchant, N. B., Levy, S. E., Coffey, R. J., and Caprioli, R. M. (2004) Proteome analysis of human colon cancer by two-dimensional difference gel electrophoresis and mass spectrometry, Proteomics 4, 793–811.PubMedCrossRefGoogle Scholar
  13. 13.
    Gade, D., Thiermann, J., Markowsky, D., and Rabus, R. (2003) Evaluation of two-dimensional difference gel electrophoresis for protein profiling. Soluble proteins of the marine bacterium Pirellula sp. strain 1, J Mol Microbiol Biotechnol 5, 240–251.PubMedCrossRefGoogle Scholar
  14. 14.
    Bollag, D., Edelstein, SJ. (1991) Protein extraction, in Protein Methods (Wiley-Liss, N., Ed.).Google Scholar
  15. 15.
    Scopes, R. (1987) Making an extract, in Protein purification: Principles and Practice (Springer Verlag, N., Ed.) 2nd ed.Google Scholar
  16. 16.
    Pennington, S. R., Wilkins, M. R., Hochstrasser, D. F., and Dunn, M. J. (1997) Proteome analysis: from protein characterization to biological function, Trends Cell Biol 7, 168–173.PubMedCrossRefGoogle Scholar
  17. 17.
    Lenstra, J. A., and Bloemendal, H. (1983) Topography of the total protein population from cultured cells upon fractionation by chemical extractions, Eur J Biochem 135, 413–423.PubMedCrossRefGoogle Scholar
  18. 18.
    Toda, T., Ishijima, Y., Matsushita, H., Yoshida, M., and Kimura, N. (1994) Detection of thymopoietin-responsive proteins in nude mouse spleen cells by two-dimensional polyacrylamide gel electrophoresis and image processing, Electrophoresis 15, 984–987.PubMedCrossRefGoogle Scholar
  19. 19.
    Cull, M., and McHenry, C. S. (1990) Preparation of extracts from prokaryotes, Methods Enzymol 182, 147–153.PubMedCrossRefGoogle Scholar
  20. 20.
    Jazwinski, S. M. (1990) Preparation of extracts from yeast, Methods Enzymol 182, 154–174.PubMedCrossRefGoogle Scholar
  21. 21.
    Kawaguchi, S., and Kuramitsu, S. (1995) Separation of heat-stable proteins from Thermus thermophilus HB8 by two-dimensional electrophoresis, Electrophoresis 16, 1060–1066.PubMedCrossRefGoogle Scholar
  22. 22.
    Teixeira-Gomes, A. P., Cloeckaert, A., Bezard, G., Dubray, G., and Zygmunt, M. S. (1997) Mapping and identification of Brucella melitensis proteins by two-dimensional electrophoresis and microsequencing, Electrophoresis 18, 156–162.PubMedCrossRefGoogle Scholar
  23. 23.
    Gorg, A., Boguth, G., Obermaier, C., Posch, A., and Weiss, W. (1995) Two-dimensional polyacrylamide gel electrophoresis with immobilized pH gradients in the first dimension (IPG-Dalt): the state of the art and the controversy of vertical versus horizontal systems, Electrophoresis 16, 1079–1086.PubMedCrossRefGoogle Scholar
  24. 24.
    Gorg, A., Postel, W., Domscheit, A., and Gunther, S. (1988) Two-dimensional electrophoresis with immobilized pH gradients of leaf proteins from barley (Hordeum vulgare): method, reproducibility and genetic aspects, Electrophoresis 9, 681–692.PubMedCrossRefGoogle Scholar
  25. 25.
    Gorg, A., Postel, W., and Gunther, S. (1988) The current state of two-dimensional electrophoresis with immobilized pH gradients, Electrophoresis 9, 531–546.PubMedCrossRefGoogle Scholar
  26. 26.
    Dignam, J. D. (1990) Preparation of extracts from higher eukaryotes, Methods Enzymol 182, 194–203.PubMedCrossRefGoogle Scholar
  27. 27.
    Blomberg, A., Blomberg, L., Norbeck, J., Fey, S. J., Larsen, P. M., Larsen, M., Roepstorff, P., Degand, H., Boutry, M., Posch, A., and et al. (1995) Interlaboratory reproducibility of yeast protein patterns analyzed by immobilized pH gradient two-dimensional gel electrophoresis, Electrophoresis 16, 1935–1945.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Dipartimento di Scienze e Tecnologie BiomedicheUniversità degli Studi di MilanoSegrateItaly

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