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 Wang

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

Abstract

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 affi nity purifi cation 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 affi nity between biotin and streptavidin, allowing for high affi nity purifi cation of protein complexes without the use of antibodies. Here, we describe an improved large-scale purifi cation of multi-protein complexes in mouse embryonic stem cells by in vivo biotinylation, complemented with standard antibody and/or FLAG based affi nity captures. This combined strategy benefi ts from the high effi ciency of the streptavidin pull-down and the validation of the most highly confi dent interacting partners through the two alternative approaches.

Original languageEnglish
Pages (from-to)135-149
Number of pages15
JournalMethods in Molecular Biology
Volume1177
DOIs
StatePublished - 2014

Keywords

  • Biotinylation
  • BirA
  • ESC
  • Mass spectrometry
  • Multi-protein complexes
  • Purification
  • Streptavidin

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