Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation
| Autor: | Jack Greenblatt, Zhen Yuan Lin, Harshika Jain, Ashley Hutchinson, Bryan Krastins, Mani Ravichandran, Anandi Bhattacharya, Lei Zhao, Alma Seitova, Tin Nguyen, Susanne Gräslund, Marcin Paduch, Anne-Claude Gingras, Gregory Byram, Cheryl H. Arrowsmith, Jeffrey R. Whiteaker, Ruedi Aebersold, Xinghua Guo, Maryann Vogelsang, Evan Dowdell, Aled M. Edwards, Ben C. Collins, Peter Loppnau, Gouri Vadali, Andrew Emili, Sadhna Phanse, Shuye Pu, Nan Zhong, Matthias Gstaiger, Hongbo Guo, Sachdev S. Sidhu, Amanda G. Paulovich, Brett Larsen, Lori Frappier, Jonathan B. Olsen, Shohei Koide, Edyta Marcon, Maria Fenner, Guoqing Zhong, Shane Miersch, Mary F. Lopez, Shoshana J. Wodak, Anthony A. Kossiakoff, Jacob J. Kennedy |
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| Rok vydání: | 2015 |
| Předmět: |
Proteomics
Proteome Immunoprecipitation Computational biology Biochemistry Mass Spectrometry Immunoglobulin G Antibody Specificity Peptide Library Escherichia coli Humans Cloning Molecular Peptide library Immunoglobulin Fragments Molecular Biology biology Antibodies Monoclonal Computational Biology Proteins Reproducibility of Results Cell Biology Gold standard (test) Molecular biology Chromatin HEK293 Cells biology.protein Antibody Standard operating procedure Biotechnology |
| Zdroj: | Nature Methods. 12:725-731 |
| ISSN: | 1548-7105 1548-7091 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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