Unit

UNIT 1B.5 Tandem Affinity Purification of Protein Complexes in Mouse Embryonic Stem Cells Using In Vivo Biotinylation

  1. Jianlong Wang1,
  2. Alan B. Cantor1,
  3. Stuart H. Orkin1,2

Published Online: 1 OCT 2009

DOI: 10.1002/9780470151808.sc01b05s11

Lab Protocol Title

Current Protocols in Stem Cell Biology

Current Protocols in Stem Cell Biology

Additional Information

How to Cite

Wang, J., Cantor, A. B. and Orkin, S. H. 2009. Tandem Affinity Purification of Protein Complexes in Mouse Embryonic Stem Cells Using In Vivo Biotinylation. Current Protocols in Stem Cell Biology. 11:B:1B.5:1B.5.1–1B.5.17.

Author Information

  1. 1

    Children's Hospital and the Dana Farber Cancer Institute, Harvard Medical School, Harvard Stem Cell Institute, Boston, Massachusetts

  2. 2

    The Howard Hughes Medical Institute, Boston, Massachusetts

Publication History

  1. Published Online: 1 OCT 2009
  2. Published Print: DEC 2009
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Abstract

Streptavidin affinity purification of protein complexes, in combination with in vivo biotinylation of critical transcription factors, has contributed to the analysis of the pluripotent state in mouse embryonic stem (ES) cells and made it possible to construct 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 an 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. Curr. Protoc. Stem Cell Biol. 11:1B.5.1-1B.5.17. © 2009 by John Wiley & Sons, Inc.

Keywords:

  • tandem affinity purification;
  • in vivo biotinylation;
  • protein-protein interaction;
  • embryonic stem cells
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