title: Analytical Ultracentrifuge Analysis of Nucleosomes Assembled from Recombinant, Acid-Extracted, HPLC-Purified Histones.
abstract: The accumulating discoveries of new posttranslational modifications (PTMs) and the increasing relevance of histone variants within the frame of epigenetics demand the availability of methods for a rapid and efficient nucleosome reconstitution to analyze their structural and functional implications. Here we describe a method suitable for this purpose, starting from bacterially expressed histones, solubilized by acid and purified by reversed-phase high-performance liquid chromatography. This method allows the preparation of micrograms to milligram amounts of in vitro-assembled nucleosomes. Finally, we demonstrate the efficiency of this method for the structural analysis of nucleosomes in the analytical ultracentrifuge.
authors.affiliation: Department of Biochemistry and Microbiology, University of Victoria, 3800 Finnerty Road, Victoria, BC, Canada, V8W 3P6.
authors.forename: Manjinder S
authors.initials: MS
authors.lastname: Cheema
authors.affiliation: Department of Biochemistry and Microbiology, University of Victoria, 3800 Finnerty Road, Victoria, BC, Canada, V8W 3P6. jausio@uvic.ca.
authors.forename: Juan
authors.initials: J
authors.lastname: Ausió
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange; D006657:Histones / Q000378:metabolism*; D006801:Humans; D009707:Nucleosomes / Q000378:metabolism*; D014461:Ultracentrifugation / Q000379:methods*
chemical_list: D006657:Histones; D009707:Nucleosomes
keywords: Acid extraction; Analytical ultracentrifuge (AUC); Fast protein liquid chromatography (FPLC); Nucleosome; Recombinant histones; Reversed-phase high-performance liquid chromatography (HPLC)