Salt-urea, sulfopropyl-sepharose, and covalent chromatography methods for histone isolation and fractionati

Abstract

The histones are essential basic proteins intimately involved in most DNA-templated processes. Thus, their purifi cation and fractionation for analysis and their use for in vitro chromatin transactions are of fundamental importance for understanding their role in chromatin structure and regulation of DNA functions. Here are described three new protocols for histone isolation from undisturbed whole cells. They avoid the conventional non-denaturing cell lysis, which affects the native posttranslational modifi cations of histones, and the cumbersome use of reverse-phase high-performance liquid chromatography. The three methodologies are shorter than the conventionally used protocols. The salt-urea method exploits the stability of the cell nucleus in salt solutions containing 8 M urea, whereas the cell cytoplasm and the majority of nuclear components, except H3/H4, are washed away. This protocol yields highly purifi ed H3/H4 in a few minutes without the use of chromatography steps. The acid extraction-s ulfo p ropyl (SP)-Sepharose protocol uses acidic solution for direct extraction of histones from undisturbed cells. Following extraction, the solution is neutralized with Tris-HCl, and run through a SP column. H2A/H2B are eluted from the SP-Sepharose at 0.8 M NaCl, with H3/H4 subsequently eluted at 2 M NaCl. This procedure yields highly purifi ed H2A/H2B and H3/H4. Finally, covalent chromatography on t hio p ropyl-S epharose (TPS) allows the separation of H3 from H4, by covalently binding H3 through its unique cysteine residue to the resin; H4 is recovered in the fl ow-through and wash fractions, and H3 is eluted with dithiothreitol. © 2014 Springer Science+Business Media, New York.