Protocol

The Proteomics Protocols Handbook

pp 37-48

Differential Detergent Fractionation of Eukaryotic Cells

  • Melinda L. RamsbyAffiliated withDepartment of Medicine, School of Medicine, University of Connecticut Health Center
  • , Gregory S. MakowskiAffiliated withDepartment of Medicine, School of Medicine, University of Connecticut Health Center

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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 (15). 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 (46). 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 (715).