Use of in vivo biotinylation to study protein-protein and protein-DNA interactions in mouse embryonic stem cells.
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 approximately 50 d to complete, and it takes another approximately 15 d and approximately 3 d to perform affinity purification of protein complexes and bioChIP, respectively.
Kim
Jonghwan
J
Division of Hematology-Oncology, Department of Pediatric Oncology, Children's Hospital and the Dana Farber Cancer Institute, Harvard Medical School, Harvard Stem Cell Institute, Boston, Massachusetts 02115, USA.
Cantor
Alan B
AB
Orkin
Stuart H
SH
Wang
Jianlong
J