Wang Jianlong, Cantor Alan B, Orkin Stuart H
Children's Hospital and the Dana Farber Cancer Institute, Harvard Medical School, Harvard Stem Cell Institute, Boston, Massachusetts, USA.
Curr Protoc Stem Cell Biol. 2009 Mar;Chapter 1:Unit1B.5. doi: 10.1002/9780470151808.sc01b05s8.
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.
在剖析小鼠胚胎干细胞（ES细胞）的多能状态时，我们采用了对关键转录因子进行体内生物素化的方法，以通过链霉亲和素亲和纯化蛋白质复合物，并构建了一个蛋白质-蛋白质相互作用网络。这有助于发现新的多能性因子，并更好地理解干细胞的多能性。在这里，我们描述了在小鼠ES细胞中建立体内生物素化系统以及使用体内生物素化亲和纯化多蛋白复合物的详细步骤。此外，我们还提供了一个方案，即在提交进行质谱（MS）蛋白质鉴定之前，采用SDS-PAGE分级分离来降低样品复杂性。
