NP-40 reduces contamination by endogenous biotinylated carboxylases during purification of biotin tagged nuclear proteins
Protein Expression and Purification , Volume 89 - Issue 1 p. 80- 83
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.
|Nuclear extracts Affinity purification Protein complexes Transcription factors BirA biotin ligase Biotin tagging|
|Protein Expression and Purification|
|Organisation||Department of Biochemistry|
Papageorgiou, D.N, Demmers, J.A.A, & Strouboulis, J. (2013). NP-40 reduces contamination by endogenous biotinylated carboxylases during purification of biotin tagged nuclear proteins. Protein Expression and Purification, 89(1), 80–83. doi:10.1016/j.pep.2013.02.015