Use of in vivo biotinylation to study protein-protein and protein-DNA interactions in mouse embryonic stem cells
Use of in vivo biotinylation to study protein-protein and protein-DNA interactions in mouse embryonic stem cells
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DOI：
10.1038/nprot.2009.23
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发表时间：
2009-01-01
期刊：
影响因子：
14.8
通讯作者：
Wang, Jianlong
中科院分区：
文献类型：
--
作者：
Kim, Jonghwan;Cantor, Alan B.;Wang, Jianlong
在基因调控中，蛋白质作为蛋白质复合物的成员来识别染色体上的靶DNA位点。在解剖小鼠胚胎干（mES）细胞的多能状态时，我们分别使用了关键转录因子的体内生物素化来亲和纯化蛋白质复合物和染色质免疫沉淀（ChIP）芯片来鉴定靶标。在这里，我们详细描述了这些研究的程序，以解剖蛋白质-蛋白质和蛋白质- dna相互作用在mES细胞。具体来说，将描述以下三个程序：(i)在mES细胞中建立体内生物素化系统；（ii）通过一步链霉亲和素捕获和串联抗flag /链霉亲和素亲和纯化对多蛋白复合物进行亲和纯化；（iii）生物素介导芯片（bioChIP）。完成系统设置大约需要50天，对蛋白复合物和bioChIP进行亲和纯化分别需要大约15天和大约3天。
In gene regulation, proteins function as members of protein complexes to recognize chromosomal target DNA loci. In dissecting the pluripotent state in mouse embryonic stem (mES) cells, we have used in vivo biotinylation of critical transcription factors for affinity purification of protein complexes and chromatin immunoprecipitation (ChIP)-on-chip for target identification, respectively. Here, we describe detailed procedures for such studies to dissect protein-protein and protein-DNA interactions in mES cells. Specifically, the following three procedures will be described: (i) in vivo biotinylation system setup in mES cells; (ii) affinity purification of multiprotein complexes by one-step streptavidin capture and tandem anti-FLAG/streptavidin affinity purification; (iii) biotin-mediated ChIP (bioChIP). The system setup takes similar to 50 d to complete, and it takes another similar to 15 d and similar to 3 d to perform affinity purification of protein complexes and bioChIP, respectively.