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

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

  • Marcon E Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.
  • Jain H Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
  • Bhattacharya A Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
  • Guo H Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.
  • Phanse S Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.
  • Pu S 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.
  • Byram G BRIMS (Biomarker Research Initiatives in MS), Thermo Fisher Scientific, Cambridge, Massachusetts, USA.
  • Collins BC Department of Biology, Institute of Molecular Systems Biology, Zurich, Switzerland.
  • Dowdell E Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA.
  • Fenner M Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
  • Guo X Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.
  • Hutchinson A Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
  • Kennedy JJ Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
  • Krastins B BRIMS (Biomarker Research Initiatives in MS), Thermo Fisher Scientific, Cambridge, Massachusetts, USA.
  • Larsen B Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
  • Lin ZY Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.
  • Lopez MF BRIMS (Biomarker Research Initiatives in MS), Thermo Fisher Scientific, Cambridge, Massachusetts, USA.
  • Loppnau P Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
  • Miersch S Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.
  • Nguyen T Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
  • Olsen JB Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
  • Paduch M Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA.
  • Ravichandran M Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
  • Seitova A Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
  • Vadali G BRIMS (Biomarker Research Initiatives in MS), Thermo Fisher Scientific, Cambridge, Massachusetts, USA.
  • Vogelsang MS BRIMS (Biomarker Research Initiatives in MS), Thermo Fisher Scientific, Cambridge, Massachusetts, USA.
  • Whiteaker JR Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
  • Zhong G Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.
  • Zhong N Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
  • Zhao L Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
  • Aebersold R 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.
  • Arrowsmith CH Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
  • Emili A 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.
  • Frappier L Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
  • Gingras AC 1] Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada. [2] Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
  • Gstaiger M 1] Department of Biology, Institute of Molecular Systems Biology, Zurich, Switzerland. [2] Competence Center for Systems Physiology and Metabolic Diseases, Zurich, Switzerland.
  • Paulovich AG Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
  • Koide S Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA.
  • Kossiakoff AA Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA.
  • Sidhu SS Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada.
  • Wodak SJ 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.
  • Gräslund S Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
  • Greenblatt JF 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.
  • Edwards AM Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
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  • 2015-06-30
Published in:
  • Nature methods. - 2015
English 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.
Language
  • English
Open access status
closed
Identifiers
Persistent URL
https://folia.unifr.ch/global/documents/35867
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