Herrmann Christin, Avgousti Daphne C, Weitzman Matthew D
Division of Cancer Pathobiology, Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, USA.
Cell & Molecular Biology Graduate Group, University of Pennsylvania, Philadelphia, USA.
Bio Protoc. 2017 Mar 20;7(6). doi: 10.21769/BioProtoc.2175.
Nucleosomes are the core units of cellular chromatin and are comprised of 147 base pairs (bp) of DNA wrapped around an octamer of histone proteins. Proteins such as chromatin remodelers, transcription factors, and DNA repair proteins interact dynamically with chromatin to regulate access to DNA, control gene transcription, and maintain genome integrity. The extent of association with chromatin changes rapidly in response to stresses, such as immune activation, oxidative stress, or viral infection, resulting in downstream effects on chromatin conformation and transcription of target genes. To elucidate changes in the composition of proteins associated with chromatin under different conditions, we adapted existing protocols to isolate nuclei and fractionate cellular chromatin using a gradient of salt concentrations. The presence of specific proteins in different salt fractions can be assessed by Western blotting or mass spectrometry, providing insight into the degree to which they are associated with chromatin.
核小体是细胞染色质的核心单位，由缠绕在组蛋白八聚体上的147个碱基对（bp）的DNA组成。诸如染色质重塑因子、转录因子和DNA修复蛋白等蛋白质与染色质动态相互作用，以调节对DNA的访问、控制基因转录并维持基因组完整性。与染色质的结合程度会随着免疫激活、氧化应激或病毒感染等应激反应而迅速变化，从而对染色质构象和靶基因转录产生下游影响。为了阐明在不同条件下与染色质相关的蛋白质组成的变化，我们采用了现有的方案，通过盐浓度梯度分离细胞核并对细胞染色质进行分级分离。不同盐分级中特定蛋白质的存在可以通过蛋白质印迹法或质谱法进行评估，从而深入了解它们与染色质的结合程度。
