Differential Salt Fractionation of Nuclei to Analyze Chromatin-associated Proteins from Cultured Mammalian Cells
Differential Salt Fractionation of Nuclei to Analyze Chromatin-associated Proteins from Cultured Mammalian Cells
复制标题
DOI：
10.21769/bioprotoc.2175
复制
发表时间：
2017-03-20
期刊：
影响因子：
0.8
通讯作者：
Weitzman, Matthew D.
中科院分区：
文献类型：
--
作者：
Herrmann, Christin;Avgousti, Daphne C.;Weitzman, Matthew D.
核小体是细胞染色质的核心单位，由147个碱基对（bp）的DNA包裹在组蛋白八聚体周围组成。蛋白质如染色质重塑因子、转录因子和DNA修复蛋白与染色质动态地相互作用以调节对DNA的接近、控制基因转录并维持基因组完整性。与染色质的关联程度响应于应激而迅速变化，诸如免疫活化、氧化应激或病毒感染，导致对染色质构象和靶基因转录的下游效应。为了阐明不同条件下与染色质相关的蛋白质组成的变化，我们采用现有的协议，使用盐浓度梯度分离细胞核和fractionalcellular染色质。不同盐组分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相关的程度。
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.