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

Edyta Marcon, Harshika Jain, Anandi Bhattacharya, Hongbo Guo, Sadhna Phanse, Shuye Pu, Gregory Byram, Ben C. Collins, Evan Dowdell, Maria Fenner, Xinghua Guo, Ashley Hutchinson, Jacob J. Kennedy, Bryan Krastins, Brett Larsen, Zhen-Yuan Lin, Mary F. Lopez, Peter Loppnau, Shane Miersch, Tin NguyenJonathan B. Olsen, Marcin Paduch, Mani Ravichandran, Alma Seitova, Gouri Vadali, Maryann S. Vogelsang, Jeffrey R. Whiteaker, Guoqing Zhong, Nan Zhong, Lei Zhao, Ruedi Aebersold, Cheryl H. Arrowsmith, Andrew Emili, Lori Frappier, Anne-Claude Gingras, Matthias Gstaiger, Amanda G. Paulovich, Shohei Koide, Anthony A. Kossiakoff, Sachdev S. Sidhu, Shoshana J. Wodak, Susanne Gräslund, Jack F. Greenblatt, Aled M. Edwards

Research output: Contribution to journalArticlepeer-review

70 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)725-731
Number of pages7
JournalNature Methods
Volume12
Issue number8
DOIs
Publication statusPublished - 29 Jun 2015

Keywords

  • Proteomics
  • Proteomic analysis
  • Protein enrichment

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