title: NP-40 reduces contamination by endogenous biotinylated carboxylases during purification of biotin tagged nuclear proteins.
abstract: We describe here a simple procedure for greatly reducing contamination of nuclear extracts by naturally biotinylated cytoplasmic carboxylases, which represent a major source of non-specific background when employing BirA-mediated biotinylation tagging for the purification and characterization of nuclear protein complexes by mass spectrometry. We show that the use of 0.5% of the non-ionic detergent Nonidet-40 (NP-40) during cell lysis and nuclei isolation is sufficient to practically eliminate contamination of nuclear extracts by carboxylases and to greatly reduce background signals in downstream mass spectrometric analyses.
authors.affiliation: Division of Molecular Oncology, Biomedical Sciences Research Center Alexander Fleming, 34 Fleming Street, Vari GR-166 72, Greece.
authors.forename: Dimitris N
authors.initials: DN
authors.lastname: Papageorgiou
authors.forename: Jeroen
authors.initials: J
authors.lastname: Demmers
authors.forename: John
authors.initials: J
authors.lastname: Strouboulis
mesh_terms: D001710:Biotin / Q000737:chemistry*; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry; D002457:Cell Extracts / Q000737:chemistry / Q000302:isolation & purification; D002467:Cell Nucleus / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry; D009687:Nuclear Proteins / Q000302:isolation & purification*; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000737:chemistry*; D012097:Repressor Proteins / Q000737:chemistry
chemical_list: D002457:Cell Extracts; D029968:Escherichia coli Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; D011092:Polyethylene Glycols; D001710:Biotin; D017830:Octoxynol; C010615:Nonidet P-40; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli