NP-40 reduces contamination by endogenous biotinylated carboxylases during purification of biotin tagged nuclear proteins

•Comparing nuclear extract methods to reduce biotinylated cytoplasmic carboxylases.•NP-40 significantly reduces nuclear contamination by carboxylases.•Mass spec shows reduced carboxylase background binding to streptavidin beads.•Method is widely applicable for nuclear protein purification by biotin tagging.

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.