Differential Detergent Fractionation of Eukaryotic Cells
Part of the
Springer Protocols Handbooks
book series (SPH)
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).
KeywordsStock 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.
Lenstra, J. A. and Bloemendal, H. (1983) Topography of the total protein population from cultured cells upon fractionation by chemical extractions. Eur. J. Biochem.
, 413–423.PubMedCrossRefGoogle Scholar
Lenk, R., Ransom, L., Kaufman, Y., and Penman, S. (1977) A cytoskeletal structure with associated polyribosomes obtained from HeLa cells. Cell
, 67–78.PubMedCrossRefGoogle Scholar
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
, 4737–4741.PubMedCrossRefGoogle Scholar
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.
, 1973–1984.PubMedCrossRefGoogle Scholar
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.
, 17–33.PubMedGoogle Scholar
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
, 559–568.PubMedCrossRefGoogle Scholar
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
, 113–120.PubMedCrossRefGoogle Scholar
Bird, R. C. and Sells, B. H. (1986) Cytoskeleton involvement in the distribution of mRNP complexes and small cytoplasmic RNAs. Biochim. Biophys. Acta
, 251–225.Google Scholar
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.
, 565–575.PubMedCrossRefGoogle Scholar
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.
, 793–800.PubMedGoogle Scholar
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.
, 1209–1218.PubMedCrossRefGoogle Scholar
Belin, M.-T. and Boulanger, P. (1985) Cytoskeletal proteins associated with intracytoplasmic human adenovirus at an early stage of infection. Exp. Cell Res.
, 356–370.PubMedCrossRefGoogle Scholar
Ciamper, F. (1988) The role of the cytoskeleton and nuclear matrix in viral replication. Acta Virol.
, 338–350.Google Scholar
Tanquay, R. M. (1983) Genetic regulation during heat shock and function of heat-shock proteins: a review. Can. J. Biochem. Cell. Biol.
, 387–394.CrossRefGoogle Scholar
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.
, 1198–1211.PubMedCrossRefGoogle Scholar
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
, 265–277.PubMedCrossRefGoogle Scholar
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.
, 3207–3219.PubMedGoogle Scholar
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
Peterson, G. L. (1983) Determination of total protein. Meth. Enzymol.
, 95–119.PubMedCrossRefGoogle Scholar
O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem.
, 4007–4021.Google Scholar
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
, 1133–1142.CrossRefGoogle Scholar
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.
, 144–145.PubMedCrossRefGoogle Scholar
Zuurendonk, P. F. and Tager, J. M. (1974) Rapid separation of particulate components and soluble cytoplasm of isolated rat-liver cells. Biochim. Biophys. Acta
, 393–399.PubMedCrossRefGoogle Scholar
Lever, M. (1977) Peroxides in detergents as interferring factors in biochemical analysis. Anal. Biochem.
, 274–284.PubMedCrossRefGoogle Scholar
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.
, 112–117.PubMedCrossRefGoogle Scholar
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.
, 270–274PubMedCrossRefGoogle Scholar
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
, 3430–3434.PubMedCrossRefGoogle Scholar
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.
, 437–449.PubMedCrossRefGoogle Scholar
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.
, 809–815.PubMedGoogle Scholar
Morgenstern, R., Meijer, J., Depierre, J. W., and Ernster, L. (1980) Characterization of ratliver microsomal glutathione-S-transferase activity. Eur. J. Biochem.
, 167–174.PubMedCrossRefGoogle Scholar
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.
, 392–411.Google Scholar
Bordier, C. (1981) Phase separation of integral membrane proteins in Triton X-114 solutions. J. Biol. Chem.
, 1604–1607.PubMedGoogle Scholar
Pryde, J. G. and Phillips, J. H. (1986) Fractionation of membrane proteins by temperatureinduced phase separation in Triton X-114. Biochem. J.
, 525–533.PubMedGoogle Scholar
Capco, D. G., Wan, K. M., and Penman, S. (1982) The nuclear matrix: three-dimensional architecture and protein composition. Cell
, 847–858.PubMedCrossRefGoogle Scholar
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.
, 345–365.PubMedCrossRefGoogle Scholar
© Humana Press Inc., Totowa, NJ 2005