Tandem affinity purification of protein complexes in mouse embryonic stem cells using in vivo biotinylation.
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.
Wang
Jianlong
J
Children's Hospital and the Dana Farber Cancer Institute, Harvard Medical School, Harvard Stem Cell Institute, Boston, Massachusetts, USA.
Cantor
Alan B
AB
Orkin
Stuart H
SH
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
D046912:Multiprotein Complexes; D014157:Transcription Factors; D019809:Streptavidin