title: An improved in vivo biotinylation strategy combined with FLAG and antibody based approaches for affinity purification of protein complexes in mouse embryonic stem cells.
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 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.
authors.affiliation: Department of Developmental and Regenerative Biology, Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, Atran Building, AB7-10D, 1428 Madison Ave, New York, NY, 10029, USA.
authors.forename: Francesco
authors.initials: F
authors.lastname: Faiola
authors.forename: Arven
authors.initials: A
authors.lastname: Saunders
authors.forename: Baoyen
authors.initials: B
authors.lastname: Dang
authors.forename: Jianlong
authors.initials: J
authors.lastname: Wang
mesh_terms: D000818:Animals; D019921:Biotinylation; D002846:Chromatography, Affinity / Q000379:methods*; D053595:Embryonic Stem Cells / Q000737:chemistry*; D051379:Mice; D008967:Molecular Biology / Q000379:methods*; D046912:Multiprotein Complexes / Q000737:chemistry / Q000302:isolation & purification*; D009842:Oligopeptides / Q000737:chemistry; D019809:Streptavidin
chemical_list: D046912:Multiprotein Complexes; D009842:Oligopeptides; D019809:Streptavidin; C085706:FLAG peptide