title: Rapid Isolation of Nuclei from Cells In Vitro.
abstract: This protocol presents a rapid, efficient, and practical (REAP) method to separate nuclei from cultured cells in vitro with as little damage and contamination as possible. The REAP procedure is performed at low temperature and takes <2 min, which minimizes protein degradation, protein modification, and diffusion of soluble proteins out of the nuclear compartment while maintaining the integrity of protein complexes. A mild detergent, NP-40, is used together with mild mechanical shearing to disrupt the plasma membrane, leaving the nuclear membrane intact. The REAP method can be used with various cell lines grown in vitro and requires minimal optimization. The isolated nuclei are suitable for numerous downstream applications (e.g., western blotting, 2D gel electrophoresis, and immunoprecipitation). If desired, aliquots of whole-cell lysate and the cytoplasmic fraction can be saved for comparison.
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; D002478:Cells, Cultured; D002498:Centrifugation / Q000379:methods*; D003080:Cold Temperature; D003902:Detergents / Q000378:metabolism; D006801:Humans; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000378:metabolism; D013997:Time Factors
chemical_list: D003902:Detergents; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40