Use of in vivo biotinylation to study protein-protein and protein-DNA interactions in mouse embryonic stem cells

被引：108

作者：

Kim, Jonghwan ^{[1
,2
,3
]}

Cantor, Alan B. ^{[1
,2
]}

Orkin, Stuart H. ^{[1
,2
,3
]}

Wang, Jianlong ^{[1
,2
]}

机构：

[1] Harvard Univ, Sch Med, Harvard Stem Cell Inst,Div Hematol Oncol, Dept Pediat Oncol,Childrens Hosp, Boston, MA 02115 USA

[2] Harvard Univ, Sch Med, Harvard Stem Cell Inst, Dana Farber Canc Inst, Boston, MA 02115 USA

[3] Howard Hughes Med Inst, Boston, MA 02115 USA

来源：

NATURE PROTOCOLS | 2009年 / 4卷 / 04期

关键词：

CHROMATIN IMMUNOPRECIPITATION; PLURIPOTENCY; PURIFICATION; COMPLEXES; NETWORK; CHIP;

D O I：

10.1038/nprot.2009.23

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页码：506 / 517

页数：12

共 18 条

[1] Polycomb complexes repress developmental regulators in murine embryonic stem cells [J].