An Improved In Vivo Biotinylation Strategy Combined with FLAG and Antibody Based Approaches for Affinity Purification of Protein Complexes in Mouse Embryonic Stem Cells

  • Francesco Faiola
  • Arven Saunders
  • Baoyen Dang
  • Jianlong WangEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1177)


The proteome in mouse embryonic stem cells has not been extensively studied in comparison to other cellular systems, limiting our understanding of multi-protein complex functions in stem cell biology. Several affinity purification techniques followed by mass spectrometry analysis have been designed and validated to identify protein–protein interaction networks. One such approach relies on in vivo biotinylation of a protein of interest and subsequent pull-down of its interacting partners using streptavidin-conjugated agarose beads. This technique takes advantage of the high affinity between biotin and streptavidin, allowing for high affinity purification of protein complexes without the use of antibodies. Here, we describe an improved large-scale purification of multi-protein complexes in mouse embryonic stem cells by in vivo biotinylation, complemented with standard antibody and/or FLAG based affinity captures. This combined strategy benefits from the high efficiency of the streptavidin pull-down and the validation of the most highly confident interacting partners through the two alternative approaches.

Key words

ESC Biotinylation Streptavidin Multi-protein complexes Purification Mass spectrometry BirA 



This research was funded by grants from the National Institutes of Health (NIH) to J.W. (1R01-GM095942) and the Empire State Stem Cell Fund through New York State Department of Health (NYSTEM) to J.W. (C026420, C028103, C028121). J.W. is also a recipient of Irma T. Hirschl and Weill-Caulier Trusts Career Scientist Award.


  1. 1.
    Evans M (2011) Discovering pluripotency: 30 years of mouse embryonic stem cells. Nat Rev Mol Cell Biol 12:680–686PubMedCrossRefGoogle Scholar
  2. 2.
    Orkin SH, Hochedlinger K (2011) Chromatin connections to pluripotency and cellular reprogramming. Cell 145:835–850PubMedCrossRefGoogle Scholar
  3. 3.
    Ohtsuka S, Dalton S (2008) Molecular and biological properties of pluripotent embryonic stem cells. Gene Ther 15:74–81PubMedCrossRefGoogle Scholar
  4. 4.
    Young RA (2011) Control of the embryonic stem cell state. Cell 144:940–954PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Thelen JJ, Miernyk JA (2012) The proteomic future: where mass spectrometry should be taking us. Biochem J 444:169–181PubMedCrossRefGoogle Scholar
  6. 6.
    Collins MO, Choudhary JS (2008) Mapping multiprotein complexes by affinity purification and mass spectrometry. Curr Opin Biotechnol 19:324–330PubMedCrossRefGoogle Scholar
  7. 7.
    Bauer A, Kuster B (2003) Affinity purification-mass spectrometry. Powerful tools for the characterization of protein complexes. Eur J Biochem 270:570–578PubMedCrossRefGoogle Scholar
  8. 8.
    Wang YL, Faiola F, Martinez E (2012) Purification of multiprotein histone acetyltransferase complexes. Methods Mol Biol 809: 427–443PubMedCrossRefGoogle Scholar
  9. 9.
    Wang J, Rao S, Chu J, Shen X, Levasseur DN, Theunissen TW, Orkin SH (2006) A protein interaction network for pluripotency of embryonic stem cells. Nature 444:364–368PubMedCrossRefGoogle Scholar
  10. 10.
    Ding J, Xu H, Faiola F, Ma'ayan A, Wang J (2012) Oct4 links multiple epigenetic pathways to the pluripotency network. Cell Res 22:155–167PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Schatz PJ (1993) Use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli. Biotechnology (N Y) 11:1138–1143CrossRefGoogle Scholar
  12. 12.
    Kim J, Cantor AB, Orkin SH, Wang J (2009) Use of in vivo biotinylation to study protein-protein and protein-DNA interactions in mouse embryonic stem cells. Nat Protoc 4:506–517PubMedCrossRefGoogle Scholar
  13. 13.
    Wang J, Cantor AB, Orkin SH (2009) Tandem affinity purification of protein complexes in mouse embryonic stem cells using in vivo biotinylation. Curr Protoc Stem Cell Biol Chapter 1, Unit1B 5Google Scholar
  14. 14.
    Niwa H, Miyazaki J, Smith AG (2000) Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 24:372–376PubMedCrossRefGoogle Scholar
  15. 15.
    Kopp JL, Ormsbee BD, Desler M, Rizzino A (2008) Small increases in the level of Sox2 trigger the differentiation of mouse embryonic stem cells. Stem Cells 26:903–911PubMedCrossRefGoogle Scholar
  16. 16.
    Costa Y, Ding J, Theunissen TW, Faiola F, Hore TA, Shliaha PV, Fidalgo M, Saunders A, Lawrence M, Dietmann S, Das S, Levasseur DN, Li Z, Xu M, Reik W, Silva JC, Wang J (2013) NANOG-dependent function of TET1 and TET2 in establishment of pluripotency. Nature 495:370–374PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Rees JS, Lowe N, Armean IM, Roote J, Johnson G, Drummond E, Spriggs H, Ryder E, Russell S, St Johnston D, Lilley KS (2011) In vivo analysis of proteomes and interactomes using parallel affinity capture (iPAC) coupled to mass spectrometry. Mol Cell Proteomics 10:M110.002386PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Francesco Faiola
    • 1
  • Arven Saunders
    • 1
  • Baoyen Dang
    • 1
  • Jianlong Wang
    • 1
    Email author
  1. 1.Department of Developmental and Regenerative Biology, Black Family Stem Cell InstituteIcahn School of Medicine at Mount SinaiNew YorkUSA

Personalised recommendations