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

Edyta Marcon¹, Harshika Jain², Anandi Bhattacharya², Hongbo Guo¹, Sadhna Phanse¹, Shuye Pu³, Gregory Byram⁴, Ben C Collins⁵, Evan Dowdell⁶, Maria Fenner², Xinghua Guo¹, Ashley Hutchinson², Jacob J Kennedy⁷, Bryan Krastins⁴, Brett Larsen⁸, Zhen-Yuan Lin⁸, Mary F Lopez⁴, Peter Loppnau², Shane Miersch¹, Tin Nguyen⁹, Jonathan B Olsen⁹, Marcin Paduch⁶, Mani Ravichandran², Alma Seitova², Gouri Vadali⁴, Maryann S Vogelsang⁴, Jeffrey R Whiteaker⁷, Guoqing Zhong¹, Nan Zhong², Lei Zhao⁷, Ruedi Aebersold¹⁰, Cheryl H Arrowsmith², Andrew Emili¹¹, Lori Frappier⁹, Anne-Claude Gingras¹², Matthias Gstaiger¹³, Amanda G Paulovich⁷, Shohei Koide⁶, Anthony A Kossiakoff⁶, Sachdev S Sidhu¹, Shoshana J Wodak¹⁴, Susanne Gräslund², Jack F Greenblatt¹¹, Aled M Edwards²

Affiliations

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.

PMID: 26121405
DOI: 10.1038/nmeth.3472

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

Edyta Marcon et al. Nat Methods. 2015 Aug.

. 2015 Aug;12(8):725-31.

doi: 10.1038/nmeth.3472. Epub 2015 Jun 29.

Authors

Affiliations

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.

PMID: 26121405
DOI: 10.1038/nmeth.3472

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.

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References

1. Cell. 2005 Sep 23;122(6):947-56 - PubMed
1. Cell. 2011 Oct 28;147(3):690-703 - PubMed
1. Nature. 2015 May 21;521(7552):274-6 - PubMed
1. Cell. 2009 Jul 23;138(2):389-403 - PubMed
1. Mol Cell Biol. 2008 Oct;28(20):6262-77 - PubMed

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

Affiliations

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

Authors

Affiliations

Abstract

References

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Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Molecular Biology Databases