Use of in vivo biotinylation to study protein–protein and protein–DNA interactions in mouse embryonic stem cells (original) (raw)

Nature Protocols volume 4, pages 506–517 (2009)Cite this article

Abstract

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 ∼50 d to complete, and it takes another ∼15 d and ∼3 d to perform affinity purification of protein complexes and bioChIP, respectively.

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Acknowledgements

We acknowledge Tyler Moran for his technical assistance in developing these protocols. This work is supported by Seed Grant from the Harvard Stem Cell Institute Cell Reprogramming Program to J.W. J.K. is a Howard Hughes Medical Institute research associate. A.B.C. is supported by NIH Grant R01 HL075705. S.H.O. is an Investigator of Howard Hughes Medical Institute.

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Authors and Affiliations

  1. 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, 02115, Massachusetts, USA
    Jonghwan Kim, Alan B Cantor, Stuart H Orkin & Jianlong Wang
  2. The Howard Hughes Medical Institute, Boston, 02115, Massachusetts, USA
    Jonghwan Kim & Stuart H Orkin

Authors

  1. Jonghwan Kim
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  2. Alan B Cantor
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  3. Stuart H Orkin
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  4. Jianlong Wang
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Correspondence toJonghwan Kim or Jianlong Wang.

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Kim, J., Cantor, A., Orkin, S. et al. Use of in vivo biotinylation to study protein–protein and protein–DNA interactions in mouse embryonic stem cells.Nat Protoc 4, 506–517 (2009). https://doi.org/10.1038/nprot.2009.23

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