title: Native gel analysis of macromolecular protein complexes in cultured mammalian cells.
abstract: Native gel electrophoresis enables separation of cellular proteins in their non-denatured state. In experiments aimed at analysing proteins in higher order or multimeric assemblies (i.e. protein complexes) it offers some advantages over rival approaches, particularly as an interface technology with mass spectrometry. Here we separated fractions from HEK293 cells by native electrophoresis in order to survey protein complexes in the cytoplasmic, nuclear and chromatin environments, finding 689 proteins distributed among 217 previously described complexes. As expected, different fractions contained distinct combinations of macromolecular complexes, with subunits of the same complex tending to co-migrate. Exceptions to this observation could often be explained by the presence of subunits shared among different complexes. We investigated one identified complex, the Polycomb Repressor Complex 2 (PRC2), in more detail following affinity purification of the EZH2 subunit. This approach resulted in the identification of all previously reported members of PRC2. Overall, this work demonstrates that the use of native gel electrophoresis as an upstream separating step is an effective approach for analysis of the components and cellular distribution of protein complexes.
authors.affiliation: School of Biomolecular and Biomedical Research, University College Dublin, Belfield, Ireland.
authors.forename: Nayla
authors.initials: N
authors.lastname: Munawar
authors.affiliation: School of Biomolecular and Biomedical Research, University College Dublin, Belfield, Ireland.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Olivero
authors.affiliation: Smurfit Institute of Genetics, Trinity College, Dublin, Ireland.
authors.forename: Emilia
authors.initials: E
authors.lastname: Jerman
authors.affiliation: School of Biomolecular and Biomedical Research, University College Dublin, Belfield, Ireland.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Doyle
authors.affiliation: Smurfit Institute of Genetics, Trinity College, Dublin, Ireland.
authors.forename: Gundula
authors.initials: G
authors.lastname: Streubel
authors.affiliation: School of Biomolecular and Biomedical Research, University College Dublin, Belfield, Ireland.
authors.forename: Kieran
authors.initials: K
authors.lastname: Wynne
authors.affiliation: Smurfit Institute of Genetics, Trinity College, Dublin, Ireland.
authors.forename: Adrian
authors.initials: A
authors.lastname: Bracken
authors.affiliation: School of Biomolecular and Biomedical Research, University College Dublin, Belfield, Ireland.
authors.forename: Gerard
authors.initials: G
authors.lastname: Cagney
mesh_terms: D002843:Chromatin / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D046912:Multiprotein Complexes / Q000032:analysis* / Q000302:isolation & purification; D063347:Native Polyacrylamide Gel Electrophoresis / Q000379:methods*; D040901:Proteomics / Q000379:methods*
chemical_list: D002843:Chromatin; D046912:Multiprotein Complexes
keywords: Blue Native Electrophoresis; Polycomb; Protein complexes; Protein networks; Protein separation; Technology