title: Nuclear Protein Transport in Digitonin Permeabilized Cells.
abstract: The high concentration of cholesterol in the plasma membrane relative to the endomembranes of eukaryotic cells allows the selective permeabilization of the plasma membrane with the glycoside digitonin leaving the intracellular membrane bound organelles intact. In this chapter, we describe the basic method to use digitonin permeabilized cells to reconstitute the transport of proteins containing nuclear localization signals into the nucleus. The assay requires only a target cell line that can be permeabilized with digitonin, a source of soluble transport factors, typically provided by the cytosol fraction of cultured cells, and a cargo protein of interest. No other specialized equipment is required other than a fluorescence microscope. The assay can be used to identify transport factors required to transport specific proteins, to study the regulation of protein transport, or to study nuclear protein transport under different conditions.
authors.affiliation: Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, West Building, Room 11-335, 303 E. Chicago Ave., Chicago, IL, 60611, USA. s-adam@northwestern.edu.
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Adam
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D004072:Digitonin / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D010539:Permeability; D021381:Protein Transport; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism
chemical_list: D009687:Nuclear Proteins; D004072:Digitonin
keywords: Digitonin permeabilization; Karyopherin/importin/nuclear transport factor; Nuclear transport; Nuclear transport reconstitution