Isolation of histones and nucleosome cores from mammalian cells.
In vitro analysis of DNA in chromatin is often important for understanding mechanisms of regulation of transcription and other processes that occur on DNA. The basic unit of chromatin is the nucleosome core, containing two copies each of the core histones H2A, H2B, H3, and H4 to form a histone octamer that wraps 145 base pairs of DNA in a left-handed superhelix. In vivo, chromatin is associated with linker histones (such as H1), which facilitate the ordered packing of nucleosomes. This linker histone-containing particle is properly termed the nucleosome (or chromatosome), while the linker histone-free particle is the nucleosome core. Pure polynucleosome cores and histones can be readily isolated from mammalian tissue culture cells. This unit describes procedures for isolation and purification of nuclei, isolation of polynucleosomes lacking linker histones from these nuclei, isolation of pure populations of mono- and dinucleosome cores from oligonucleosome fractions, and isolation of core histones from purified nuclei. The methods presented here do not denature the histones, and may yield histones that are more active for in vitro assemblies.
Schnitzler
G R
GR
Tufts University School of Medicine, Boston, Massachusetts, USA.
D000818:Animals; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000737:chemistry*; D002478:Cells, Cultured; D002845:Chromatography / Q000379:methods; D004247:DNA / Q000302:isolation & purification*; D017886:Durapatite; D006657:Histones / Q000302:isolation & purification*; D006801:Humans; D009690:Nucleic Acid Conformation; D009707:Nucleosomes / Q000737:chemistry*
D006657:Histones; D009707:Nucleosomes; D004247:DNA; D017886:Durapatite