Extraction, purification and analysis of histones英文文献.pdfVIP

PROTOCOL Extraction, purification and analysis of histones 1 1 1 1,2 David Shechter , Holger L Dormann , C David Allis Sandra B Hake 1The Laboratory of Chromatin Biology, The Rockefeller University, New York, NY, USA. 2Present address: Department for Molecular Biology, Adolf-Butenandt Institute, Ludwig-Maximilians-University, Schillerstrasse 44, 80336 Munich, Germany. Correspondence should be addressed to S.B.H. (sandra.hake@med.uni-muenchen.de). Published online 7 June 2007; doi:10.1038/nprot.2007.202 Histone proteins are the major protein components of chromatin, the physiologically relevant form of the genome (or epigenome) in all eukaryotic cells. Chromatin is the substrate of many biological processes, such as gene regulation and transcription, replication, mitosis and apoptosis. Since histones are extensively post-translationally modified, the identification of these covalent marks on s l canonical and variant histones is crucial for the understanding of their biological significance. Many different biochemical techniques o c have been developed to purify and separate histone proteins. Here, we present standard protocols for acid extraction and salt o t o extraction of histones from chromatin; separation of extracted histones by reversed-phase HPLC; analysis of histones and their r p e specific post-translational modification profiles by acid urea (AU) gel electrophoresis and the additional separation of non-canonical r u t histone variants by triton AU(TAU) and 2D TAU electrophoresis; and immunoblotting of isolated histone proteins with modification- a n / speci