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

Dimitris N Papageorgiou; Jeroen Demmers; John Strouboulis

doi:10.1016/j.pep.2013.02.015

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

Dimitris N Papageorgiou, Jeroen Demmers, John Strouboulis

Comprehensive Cancer Centre

Biomedical Sciences Research Center Alexander Fleming

Research output: Contribution to journal › Article › peer-review

13 Citations (Scopus)

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.

Original language	English
Pages (from-to)	80-3
Number of pages	4
Journal	Protein Expression and Purification
Volume	89
Issue number	1
DOIs	https://doi.org/10.1016/j.pep.2013.02.015
Publication status	Published - May 2013

Keywords

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

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10.1016/j.pep.2013.02.015

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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.",

keywords = "Biotin/chemistry, Biotinylation, Carbon-Nitrogen Ligases/chemistry, Cell Extracts/chemistry, Cell Nucleus/chemistry, Escherichia coli Proteins/chemistry, Nuclear Proteins/isolation & purification, Octoxynol, Polyethylene Glycols/chemistry, Repressor Proteins/chemistry",

author = "Papageorgiou, {Dimitris N} and Jeroen Demmers and John Strouboulis",

year = "2013",

month = may,

doi = "10.1016/j.pep.2013.02.015",

language = "English",

volume = "89",

pages = "80--3",

journal = "Protein Expression and Purification",

issn = "1046-5928",

publisher = "ACADEMIC PRESS INC",

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TY - JOUR

T1 - NP-40 reduces contamination by endogenous biotinylated carboxylases during purification of biotin tagged nuclear proteins

AU - Papageorgiou, Dimitris N

AU - Demmers, Jeroen

AU - Strouboulis, John

PY - 2013/5

Y1 - 2013/5

N2 - 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.

AB - 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.

KW - Biotin/chemistry

KW - Biotinylation

KW - Carbon-Nitrogen Ligases/chemistry

KW - Cell Extracts/chemistry

KW - Cell Nucleus/chemistry

KW - Escherichia coli Proteins/chemistry

KW - Nuclear Proteins/isolation & purification

KW - Octoxynol

KW - Polyethylene Glycols/chemistry

KW - Repressor Proteins/chemistry

U2 - 10.1016/j.pep.2013.02.015

DO - 10.1016/j.pep.2013.02.015

M3 - Article

C2 - 23500724

SN - 1046-5928

VL - 89

SP - 80

EP - 83

JO - Protein Expression and Purification

JF - Protein Expression and Purification

IS - 1

ER -

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

Abstract

Keywords

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