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Differential Detergent Fractionation of Eukaryotic Cells

  • Melinda L. Ramsby
  • Gregory S. Makowski
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

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, 2, 3, 4, 5). Relative to cell fractionation by differential pelleting, DDF has the advantage of preserving the integrity of microfilament and intermediatefilament cytoskeletal networks, and is especially applicable to use with limited quantities of biomaterial (4, 5, 6). In addition, DDF is simple, highly reproducible, labor sparing, and ultracentrifuge independent. DDF is appropriate for a variety of investigations, including those aiming to: (1) enhance the delectability of low-abundance species or semi-purify components of known subcellular localization; (2) define the subcellular localization of enzymes, regulatory, or structural proteins as well as nonprotein metabolites; (3) monitor physiologic fluxes and compartmental redistribution of biomolecules under basal and stimulated conditions; (4) identify cytoskeletal-associated and interacting proteins; and (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 (7, 8, 9, 10, 11, 12, 13, 14, 15).

Keywords

Stock Buffer Solubilization Buffer Nuclear Matrix Protein Triton Extraction Staircase Pattern 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 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.PubMedCrossRefGoogle Scholar
  2. 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.PubMedCrossRefGoogle Scholar
  3. 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.PubMedCrossRefGoogle Scholar
  4. 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.PubMedCrossRefGoogle Scholar
  5. 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.PubMedGoogle Scholar
  6. 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.PubMedCrossRefGoogle Scholar
  7. 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.PubMedCrossRefGoogle Scholar
  8. 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. 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.PubMedCrossRefGoogle Scholar
  10. 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.PubMedGoogle Scholar
  11. 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.PubMedCrossRefGoogle Scholar
  12. 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.PubMedCrossRefGoogle Scholar
  13. 13.
    Ciamper, F. (1988) The role of the cytoskeleton and nuclear matrix in viral replication. Acta Virol. 170, 338–350.Google Scholar
  14. 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.CrossRefGoogle Scholar
  15. 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.PubMedCrossRefGoogle Scholar
  16. 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.PubMedCrossRefGoogle Scholar
  17. 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.PubMedGoogle Scholar
  18. 18.
    Ramsby, M. L. and Makowski, G. S. (2003) Differential detergent fractionation of eukaryotic cells and additional protocols-precipitation of tubulins and MAPs (microtubule-associated proteins) using magnesium and isolation of RNA from detergent extracts. In Simpson, R. J. (ed.), Proteins and Proteomics: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY: 126–137.Google Scholar
  19. 19.
    Peterson, G. L. (1983) Determination of total protein. Meth. Enzymol. 91, 95–119.PubMedCrossRefGoogle Scholar
  20. 20.
    O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021.Google Scholar
  21. 21.
    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.CrossRefGoogle Scholar
  22. 22.
    Duncan, R. and Hershey, J. W. B. (1984) Evaluation of isoelectric focusing running conditions during two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis: variation of gel patterns with changing conditions and optimal isoelectric focusing conditions. Anal. Biochem. 138, 144–145.PubMedCrossRefGoogle Scholar
  23. 23.
    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.PubMedCrossRefGoogle Scholar
  24. 24.
    Lever, M. (1977) Peroxides in detergents as interferring factors in biochemical analysis. Anal. Biochem. 83, 274–284.PubMedCrossRefGoogle Scholar
  25. 25.
    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.PubMedCrossRefGoogle Scholar
  26. 26.
    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, 270–274PubMedCrossRefGoogle Scholar
  27. 27.
    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.PubMedCrossRefGoogle Scholar
  28. 28.
    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.PubMedCrossRefGoogle Scholar
  29. 29.
    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.PubMedGoogle Scholar
  30. 30.
    Morgenstern, R., Meijer, J., Depierre, J. W., and Ernster, L. (1980) Characterization of ratliver microsomal glutathione-S-transferase activity. Eur. J. Biochem. 104, 167–174.PubMedCrossRefGoogle Scholar
  31. 31.
    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.Google Scholar
  32. 32.
    Bordier, C. (1981) Phase separation of integral membrane proteins in Triton X-114 solutions. J. Biol. Chem. 256, 1604–1607.PubMedGoogle Scholar
  33. 33.
    Pryde, J. G. and Phillips, J. H. (1986) Fractionation of membrane proteins by temperatureinduced phase separation in Triton X-114. Biochem. J. 233, 525–533.PubMedGoogle Scholar
  34. 34.
    Capco, D. G., Wan, K. M., and Penman, S. (1982) The nuclear matrix: three-dimensional architecture and protein composition. Cell 29, 847–858.PubMedCrossRefGoogle Scholar
  35. 35.
    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.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2005

Authors and Affiliations

  • Melinda L. Ramsby
    • 1
  • Gregory S. Makowski
    • 1
  1. 1.Department of Medicine, School of MedicineUniversity of Connecticut Health CenterFarmington

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