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Nat Methods. 2015 Aug;12(8):725-31. doi: 10.1038/nmeth.3472. Epub 2015 Jun 29.

Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation.

Author information

1
Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.
2
Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
3
1] Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada. [2] Molecular Structure and Function Program, Hospital for Sick Children, Toronto, Ontario, Canada.
4
BRIMS (Biomarker Research Initiatives in MS), Thermo Fisher Scientific, Cambridge, Massachusetts, USA.
5
Department of Biology, Institute of Molecular Systems Biology, Zurich, Switzerland.
6
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA.
7
Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
8
Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
9
Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
10
1] Department of Biology, Institute of Molecular Systems Biology, Zurich, Switzerland. [2] Competence Center for Systems Physiology and Metabolic Diseases, Zurich, Switzerland. [3] Faculty of Science, University of Zurich, Zurich, Switzerland.
11
1] Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada. [2] Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
12
1] Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada. [2] Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
13
1] Department of Biology, Institute of Molecular Systems Biology, Zurich, Switzerland. [2] Competence Center for Systems Physiology and Metabolic Diseases, Zurich, Switzerland.
14
1] Molecular Structure and Function Program, Hospital for Sick Children, Toronto, Ontario, Canada. [2] Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada. [3] Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.

Abstract

Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.

PMID:
26121405
DOI:
10.1038/nmeth.3472
[Indexed for MEDLINE]

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