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
dx.doi.org/10.1016/j.pep.2013.02.015, hdl.handle.net/1765/66384
Protein Expression and Purification
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