title: Isolation of Nuclei.
abstract: The isolation of nuclei is often the first step in studying processes such as nuclear-cytoplasmic shuttling, subcellular localization of proteins, and protein-chromatin or nuclear protein-protein interactions in response to diverse stimuli. Therefore, rapidly obtaining nuclei from cells with relatively high purity and minimal subcellular contamination, protein degradation, or postharvesting modification is highly desirable. Historically, the isolation of nuclei involved a homogenization step followed by centrifugation through high-density glycerol or sucrose. Although clean nuclei with little cytoplasmic contamination can be prepared using this method, it is typically time consuming and can allow protein degradation, protein modification, and leaching of components from the nuclei to occur. We have developed a rapid and simple fractionation method that is based on the selective dissolution of the cytoplasmic membrane (but not the nuclear membrane) using a low concentration of a nonionic detergent and rapid centrifugation steps. Here we describe important considerations when isolating nuclei from cells, introduce our rapid method, and compare this method to a more traditional protocol for isolating nuclei, noting the strengths and limitations of each approach.
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada;
authors.forename: Arash
authors.initials: A
authors.lastname: Nabbi
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada; Department of Oncology, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
authors.forename: Karl
authors.initials: K
authors.lastname: Riabowol
mesh_terms: D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002467:Cell Nucleus; D002498:Centrifugation / Q000379:methods*; D003902:Detergents / Q000378:metabolism; D006801:Humans; D013997:Time Factors
chemical_list: D003902:Detergents