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.
- Carbon-Nitrogen Ligases/chemistry
- Cell Extracts/chemistry
- Cell Nucleus/chemistry
- Escherichia coli Proteins/chemistry
- Nuclear Proteins/isolation & purification
- Polyethylene Glycols/chemistry
- Repressor Proteins/chemistry