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:
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1.
Enhance the delectability of low-abundance species or semipurify components of known subcellular localization.
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2.
Define the subcellular localization of enzymes, regulatory, or structural proteins as well as nonprotein metabolites.
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3.
Monitor physiologic fluxes and compartmental redistribution of biomolecules under basal and stimulated conditions.
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4.
Identify cytoskeletal-associated and interacting proteins.
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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
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.
Lenk, R., Ransom, L., Kaufman, Y., and Penman, S. (1977) A cytoskeletal structure with associated polyribosomes obtained from HeLa cells. Cell 10, 67–78.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Ciamper, F. (1988) The role of the cytoskeleton and nuclear matrix in viral replication. Acta Virol. 170, 338–350.
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.
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.
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.
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.
Peterson, G. L. (1983) Determination of total protein. Methods Enzymol. 91, 95–119.
O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021.
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.
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.
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.
Lever, M. (1977) Peroxides in detergents as interferring factors in biochemical analysis. Anal. Biochem. 83, 274–284.
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.
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
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.
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.
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.
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.
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.
Bordier, C. (1981) Phase separation of integral membrane proteins in Triton X-114 solutions. J. Biol. Chem. 256, 1604–1607.
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.
Capco, D. G., Wan, K. M., and Penman, S. (1982) The nuclear matrix: three-dimensional architecture and protein composition. Cell 29, 847–858.
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.
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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
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