Shechter David, Dormann Holger L, Allis C David, Hake Sandra B
The Laboratory of Chromatin Biology, The Rockefeller University, New York, NY, USA.
Nat Protoc. 2007;2(6):1445-57. 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 canonical and variant histones is crucial for the understanding of their biological significance. Many different biochemical techniques have been developed to purify and separate histone proteins. Here, we present standard protocols for acid extraction and salt extraction of histones from chromatin; separation of extracted histones by reversed-phase HPLC; analysis of histones and their specific post-translational modification profiles by acid urea (AU) gel electrophoresis and the additional separation of non-canonical histone variants by triton AU(TAU) and 2D TAU electrophoresis; and immunoblotting of isolated histone proteins with modification-specific antibodies.
组蛋白是染色质的主要蛋白质成分，染色质是所有真核细胞中基因组（或表观基因组）的生理相关形式。染色质是许多生物学过程的底物，如基因调控与转录、复制、有丝分裂和细胞凋亡。由于组蛋白存在广泛的翻译后修饰，因此鉴定常规组蛋白和变体组蛋白上的这些共价标记对于理解其生物学意义至关重要。已经开发了许多不同的生化技术来纯化和分离组蛋白。在这里，我们展示了从染色质中酸提取和盐提取组蛋白的标准方案；通过反相高效液相色谱分离提取的组蛋白；通过酸性尿素（AU）凝胶电泳分析组蛋白及其特定的翻译后修饰谱，并通过Triton AU（TAU）和二维TAU电泳进一步分离非经典组蛋白变体；以及用修饰特异性抗体对分离的组蛋白进行免疫印迹分析。
