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2-D Proteome Analysis Protocols pp 53–66Cite as

Differential Detergent Fractionation of Eukaryotic Cells

Analysis by Two-Dimensional Gel Electrophoresis

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 112))

Abstract

Differential detergent fractionation (DDF) represents an alternative method for cell fractionation that employs sequential extraction of cells or tissues with detergent-containing buffers to partition cellular proteins into structurally and functionally intact and distinct compartments (1–5). Relative to cell fractionation by differential pelleting, DDF has the advantage of preserving the integrity of microfilament and intermediate filament cytoskeletal networks, and is especially applicable to use with limited quantities of biomaterial (4–6). In addition, DDF is simple, highly reproducible, labor-sparing, and ultracentrifuge-independent. DDF is appropriate for a variety of investigations (7–15), including those objecting to:

  1. 1.

    Enhance the delectability of low-abundance species or semipurify components of known subcellular localization.

  2. 2.

    Define the subcellular localization of enzymes, regulatory, or structural proteins as well as nonprotein metabolites.

  3. 3.

    Monitor physiologic fluxes and compartmental redistribution of biomolecules under basal and stimulated conditions.

  4. 4.

    Identify cytoskeletal-associated and interacting proteins.

  5. 5.

    Investigate the role of cytoskeletal networks in the subcellular localization of endogenous and exogenous factors, including mRNA, viral components, and heat shock proteins, interactions relevant to understanding mechanisms of infection, protein turnover, and the stress response.

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References

  1. 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.

    Article  PubMed  CAS  Google Scholar 

  2. Lenk, R., Ransom, L., Kaufman, Y., and Penman, S. (1977) A cytoskeletal structure with associated polyribosomes obtained from HeLa cells. Cell 10, 67–78.

    Article  PubMed  CAS  Google Scholar 

  3. Reiter, T. and Penman, S. (1983) “Prompt” heat shock proteins: translationally-regulated synthesis of new proteins associated with nuclear matrix-intermediate filaments as an early response to heat shock. Proc. Natl. Acad. Sci. USA 80, 4737–4741.

    Article  PubMed  CAS  Google Scholar 

  4. Fey, E. G., Wan, K. M., and Penman, S. (1984) Epithelial cytoskeletal framework and nuclear matrix-intermediate filament scaffold: three-dimensional organization and protein composition. J. Cell Biol. 98, 1973–1984.

    Article  PubMed  CAS  Google Scholar 

  5. Reiter, T., Penman, S., and Capco, D. G. (1985) Shape-dependent regulation of cytoskeletal protein synthesis in anchorage-dependent and anchorage-independent cells. J. Cell Sci. 76, 17–33.

    PubMed  CAS  Google Scholar 

  6. Katsuma, Y., Marveau, N., Ohta, M., and French, S. W. (1988) Cytokeratin intermediate filaments of rat hepatocytes: different cytoskeletal domains and their three-dimensional structure. Hepatology 8, 559–568.

    Article  PubMed  CAS  Google Scholar 

  7. Cervera, M., Dreyfuss, G., and Penman, S. (1981) Messenger RNA is translated when associated with the cytoskeletal framework in normal and VSV-infected cells. Cell 23, 113–120.

    Article  PubMed  CAS  Google Scholar 

  8. Bird, R. C. and Sells, B. H. (1986) Cytoskeleton involvement in the distribution of mRNP complexes and small cytoplasmic RNAs. Biochim. Biophys. Acta 868, 251–225.

    Google Scholar 

  9. Bag, J. and Pramamik, S. (1987) Attachment of mRNA to the cytoskeletal framework and translational control of gene expression in rat L6 muscle cells. Biochem. Cell. Biol. 65, 565–575.

    Article  PubMed  CAS  Google Scholar 

  10. Doherty, F. J., Wassell, J. A., and Mayer, R. J. (1987) A putative protein sequestration site involving intermediate filaments for protein degradation by autophagy. Studies with microinjected purified glycolytic enzymes in 3T3-L1 cells. Biochem. J. 241, 793–800.

    PubMed  CAS  Google Scholar 

  11. Bonneau, A.-M., Darveau, A., and Sonenberg, N. (1985) The effect of viral infection on host protein synthesis and mRNA association with the cytoplasmic cytoskeletal structure. J. Cell Biol. 100, 1209–1218.

    Article  PubMed  CAS  Google Scholar 

  12. Belin, M.-T. and Boulanger, P. (1985) Cytoskeletal proteins associated with intracytoplasmic human adenovirus at an early stage of infection. Exp. Cell Res. 160, 356–370.

    Article  PubMed  CAS  Google Scholar 

  13. Ciamper, F. (1988) The role of the cytoskeleton and nuclear matrix in viral replication. Acta Virol. 170, 338–350.

    Google Scholar 

  14. Tanquay, R. M. (1983) Genetic regulation during heat shock and function of heat-shock proteins: a review. Can. J. Biochem. Cell. Biol. 61, 387–394.

    Article  Google Scholar 

  15. Welch, W. J. and Suhan, J. P. (1985) Morphological study of the mammalian stress response: characterization of changes in cytoplasmic organelles, cytoskeleton, and nucleoli, and appearance of intranuclear actin filament in rat fibroblasts after heat shock. J. Cell Biol. 101, 1198–1211.

    Article  PubMed  CAS  Google Scholar 

  16. Ramsby, M. L., Makowski, G. S., and Khairallah, E. A. (1994) Differential detergent fractionation of isolated hepatocytes: biochemical, immunochemical and two-dimensional gel electrophoresis characterization of cytoskeletal and noncytoskeletal compartments. Electrophoresis 15, 265–277.

    Article  PubMed  CAS  Google Scholar 

  17. Ramsby, M. L. and Kreutzer, D. L. (1993) Fibrin induction of tissue plasminogen activator expression in corneal endothelial cells in vitro. Invest. Ophthalmol. Vis. Sci. 34, 3207–3219.

    PubMed  CAS  Google Scholar 

  18. Peterson, G. L. (1983) Determination of total protein. Methods Enzymol. 91, 95–119.

    Article  PubMed  CAS  Google Scholar 

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

    Google Scholar 

  20. O’Farrell, P. Z., Goodman, H. M., and O’Farrell, P. H. (1977) High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12, 1133–1142.

    Article  Google Scholar 

  21. Duncan, R. and Hershey, J. W. B. (1984) Evaluation of isoelectric focusing running conditions during two-dimensional isoelectric focusing/sodium dodecyl sul-fate-polyacrylamide gel electrophoresis: variation of gel patterns with changing conditions and optimal isoelectric focusing conditions. Anal. Biochem. 138, 144, 145.

    Article  PubMed  CAS  Google Scholar 

  22. Zuurendonk, P. F. and Tager, J. M. (1974) Rapid separation of particulate components and soluble cytoplasm of isolated rat-liver cells. Biochim. Biophys. Acta 333, 393–399.

    Article  PubMed  CAS  Google Scholar 

  23. Lever, M. (1977) Peroxides in detergents as interferring factors in biochemical analysis. Anal. Biochem. 83, 274–284.

    Article  PubMed  CAS  Google Scholar 

  24. Chang, H. W. and Bock, E. (1980) Pitfalls in the use of commercial nonionic detergents for the solubilization of integral membrane proteins: sulfhydryl oxidizing contaminants and their elimination. Anal. Biochem. 104, 112–117.

    Article  PubMed  CAS  Google Scholar 

  25. Mackall, J., Meredith, M., and Lane, L. M. (1979) A mild procedure for the rapid release of cytoplasmic enzymes from cultured animal cells. Anal. Biochem. 95, 270–274

    Article  PubMed  CAS  Google Scholar 

  26. Fiskum, G., Craig, S. W., Decker, G. L., and Lehninger, A. L. (1980) The cytoskeleton of digitonin-treated rat hepatocytes. Proc. Natl Acad. Sci. USA 77, 3430–3434.

    Article  PubMed  CAS  Google Scholar 

  27. Weigel, P. H., Ray, D. A., and Oka, J. A. (1983) Quantitation of intracellular membrane-bound enzymes and receptors in digitonin-permeabilized cells. Anal. Biochem. 133, 437–449.

    Article  PubMed  CAS  Google Scholar 

  28. Earl, R. T., Mangiapane, E. H., Billett, E. E., and Mayer, R. J. (1987) A putative protein sequestration site involving intermediate filaments for protein degradation by autophagy. Studies with transplanted Sendai-viral envelope proteins in HTC cells. Biochem. J. 241, 809–815.

    PubMed  CAS  Google Scholar 

  29. Morgenstern, R., Meijer, J., Depierre, J. W., and Ernster, L. (1980) Characterization of rat-liver microsomal glutathione-S-transferase activity. Eur. J. Biochem. 104, 167–174.

    Article  PubMed  CAS  Google Scholar 

  30. Franke, W. W., Schmid, E., Osborn, M., and Weber, K. (1978) The intermediate-sized filaments in rat kangaroo PtK2 cells. II. Structure and composition of isolated filaments. Cytobiol. Eur. J. Cell Biol. 17, 392–411.

    CAS  Google Scholar 

  31. Bordier, C. (1981) Phase separation of integral membrane proteins in Triton X-114 solutions. J. Biol. Chem. 256, 1604–1607.

    PubMed  CAS  Google Scholar 

  32. Pryde, J. G. and Phillips, J. H. (1986) Fractionation of membrane proteins by temperature-induced phase separation in Triton X-114. Biochem. J. 233, 525–533.

    PubMed  CAS  Google Scholar 

  33. Capco, D. G., Wan, K. M., and Penman, S. (1982) The nuclear matrix: three-dimensional architecture and protein composition. Cell 29, 847–858.

    Article  PubMed  CAS  Google Scholar 

  34. Franke, W. W., Mayer, D., Schmid, E., Denk, H., and Borenfreund, E. (1981) Differences of expression of cytoskeletal proteins in cultured rat hepatocytes and hepatoma cells. Exp. Cell Res. 134, 345–365.

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. School of Medicine, University of Connecticut Health Center, Farmington, CT

    Melinda L. Ramsby

  2. Department of Laboratory Medicine, University of Connecticut Health Center, Farmington, CT

    Gregory S. Makowski

Authors
  1. Melinda L. Ramsby
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  2. Gregory S. Makowski
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Editor information

Editors and Affiliations

  1. Department of Molecular Biotechnology, University of Washington, Seattle, WA

    Andrew J. Link

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© 1999 Humana Press Inc., Totowa, NJ

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Ramsby, M.L., Makowski, G.S. (1999). Differential Detergent Fractionation of Eukaryotic Cells. In: Link, A.J. (eds) 2-D Proteome Analysis Protocols. Methods in Molecular Biology, vol 112. Humana Press. https://doi.org/10.1385/1-59259-584-7:53

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  • DOI: https://doi.org/10.1385/1-59259-584-7:53

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-524-9

  • Online ISBN: 978-1-59259-584-6

  • eBook Packages: Springer Protocols

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