PDF(Pubmed)
Abstract：
Single-molecule fluorescence microscopy (SMFM) has been shown to be informative in understanding the interaction of chromatin-associated factors with nucleosomes, the basic building unit of chromatin. Here, we present a protocol for preparing doubly labeled fluorescent nucleosomes for SMFM. We describe steps for over-expression in E. coli and purification of recombinant human core histones. We then detail fluorescent labeling of histones and nucleosomal double-stranded DNA followed by octamer refolding and nucleosome reconstitution.
摘要：
单分子荧光显微镜（SMFM）已被证明在理解染色质相关因子与核小体的相互作用方面是有益的，染色质的基本结构单元。这里,我们提出了一种用于制备SMFM的双标记荧光核小体的方案。我们描述了在大肠杆菌中过表达和纯化重组人核心组蛋白的步骤。然后，我们详细介绍了组蛋白和核小体双链DNA的荧光标记，然后进行八聚体重折叠和核小体重建。
