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NOV 08, 2016 6:00 AM PST

New Approaches to Antibody Validation Using Immunoprecipitation and Mass Spectrometry

Sponsored by: Thermo Fisher Scientific
Speaker
  • Senior R&D Manager, Mass Spectrometry Reagents, Thermo Scientific Pierce Protein Research
    Biography
      John Rogers is a Senior R&D Manager at Thermo Scientific where he manages the development of new reagents and kits for protein mass spectrometry research. John has an undergraduate degree in Biochemistry and Computer Science, and a Ph.D in Pharmacology from the University of Washington. John managed a bioinformatics group at Pfizer and a proteomics group at Abbott before joining Thermo Fisher Scientific in 2007. Since joining Thermo, John has led the development of new MS standards and calibrants, protein sample preparation reagents, and reagents for quantitative proteomic analysis, including Tandem Mass Tag reagents.

    Abstract

    Antibodies are used in a broad range of research and diagnostic applications for the enrichment, detection, and quantitation of proteins and their modifications.  Hundreds of thousands of antibodies are commercially available against thousands of proteins and their modifications. Unfortunately, many antibodies are poorly characterized, resulting in wasted time and cost as well as potentially flawed research conclusions.  To verify the performance and specificity of Thermo Scientific antibodies, we have created a comprehensive workflow to assess antibody specificity using immunoprecipitation combined with mass spectrometry (IP-MS).  In preliminary experiments, we screened more than 500 antibodies to nearly 100 key cancer signaling proteins expressed across 12 cultured tumor cell lines.  Approximately 70% of antibodies previously validated for immunocapture could be used to capture and identify the intended target, interacting proteins, and off-targets, and ~40% of antibodies not previously validated for IP were positive by IP-MS. To demonstrate the efficacy of these antibodies, we used a set of these antibodies to simultaneously immunocapture twelve proteins in the Akt/mTOR pathway, and then quantified the proteins and their phosphorylation in four IGF-stimulated cell lines using MS-based targeted quantification.  Benchmarking of these multiplexed IP-MS assays showed moderate correlation to quantitation with more traditional Western blotting, ELISA, and Luminex assays.


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