title: Tandem affinity purification of protein complexes in mouse embryonic stem cells using in vivo biotinylation.
abstract: In dissecting the pluripotent state in mouse embryonic stem (ES) cells, we have employed in vivo biotinylation of critical transcription factors for streptavidin affinity purification of protein complexes and constructed 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 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.
authors.affiliation: Children's Hospital and the Dana Farber Cancer Institute, Harvard Medical School, Harvard Stem Cell Institute, Boston, Massachusetts, USA.
authors.forename: Jianlong
authors.initials: J
authors.lastname: Wang
authors.forename: Alan B
authors.initials: AB
authors.lastname: Cantor
authors.forename: Stuart H
authors.initials: SH
authors.lastname: Orkin
mesh_terms: D000818:Animals; D019921:Biotinylation / Q000379:methods*; D002846:Chromatography, Affinity; D053595:Embryonic Stem Cells / Q000737:chemistry*; D051379:Mice; D046912:Multiprotein Complexes / Q000302:isolation & purification*; D039904:Pluripotent Stem Cells; D040901:Proteomics / Q000379:methods*; D019809:Streptavidin; D014157:Transcription Factors
chemical_list: D046912:Multiprotein Complexes; D014157:Transcription Factors; D019809:Streptavidin