Nuclear protein transport in digitonin permeabilized cells

Research output: Chapter in Book/Report/Conference proceedingChapter

4 Scopus citations

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 fl uorescence 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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc
Pages479-487
Number of pages9
DOIs
StatePublished - Jan 1 2016

Publication series

NameMethods in Molecular Biology
Volume1411
ISSN (Print)1064-3745

Keywords

  • Digitonin permeabilization
  • Karyopherin/importin/nuclear transport factor
  • Nuclear transport
  • Nuclear transport reconstitution

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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