Tandem affinity purification of protein complexes in mouse embryonic stem cells using in vivo biotinylation.
Tandem affinity purification of protein complexes in mouse embryonic stem cells using in vivo biotinylation.
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DOI：
10.1002/9780470151808.sc01b05s8
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发表时间：
2009-03-01
影响因子：
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通讯作者：
Orkin, Stuart H
中科院分区：
文献类型：
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作者：
Wang, Jianlong;Cantor, Alan B;Orkin, Stuart H
在解剖小鼠胚胎干细胞的多能状态时，我们利用体内生物素化关键转录因子纯化链霉亲和素蛋白复合物，并构建了蛋白-蛋白相互作用网络。这有助于发现新的多能性因子和更好地理解干细胞多能性。在这里，我们详细描述了在小鼠胚胎干细胞中建立体内生物素化系统的过程，以及使用体内生物素化的多蛋白复合物的亲和纯化。此外，我们提出了一种采用SDS-PAGE分离的方案，以减少提交质谱（MS）蛋白质鉴定之前的样品复杂性。
In dissecting the pluripotent state in mouse embryonic stem (ES) cells, we have employed in vivo biotinylation of critical transcription factors for streptavidin affinity purification of protein complexes and constructed a protein-protein interaction network. This has facilitated discovery of novel pluripotency factors and a better understanding of stem cell pluripotency. Here we describe detailed procedures for in vivo biotinylation system setup in mouse ES cells, and affinity purification of multi-protein complexes using in vivo biotinylation. In addition, we present a protocol employing SDS-PAGE fractionation to reduce sample complexity prior to submission for mass spectrometry (MS) protein identification.