MENU
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.


    Show Resources
    You May Also Like
    SEP 10, 2020 9:00 AM PDT
    C.E. CREDITS
    SEP 10, 2020 9:00 AM PDT
    Date: September 10, 2020 Time: 9:00am (PDT), 12:00pm (EDT) Osmolality testing is relevant throughout the entire bioprocessing workflow. As customers look to refine mAb and gene therapy workf...
    APR 07, 2020 8:00 AM PDT
    C.E. CREDITS
    APR 07, 2020 8:00 AM PDT
    DATE: April 7, 2020 TIME: 8:00am PT, 11:00am ET This webinar sets out to establish why quality control is key to robust, reliable, reproducible science. We will look at best practice criteri...
    SEP 03, 2020 9:00 AM PDT
    C.E. CREDITS
    SEP 03, 2020 9:00 AM PDT
    DATE: September 3, 2020 TIME: 09:00am PT, 12:00pm ET xxx Learning Objectives: xxx Webinars will be available for unlimited on-demand viewing after live event. LabRoots is approved as a provi...
    JUN 17, 2020 1:30 PM PDT
    C.E. CREDITS
    JUN 17, 2020 1:30 PM PDT
    Understanding the complex interplay between a pathogen and the host response is important to developing effective vaccines and therapeutics. The nCounter® Analysis System and GeoMx®...
    AUG 27, 2020 7:00 AM PDT
    C.E. CREDITS
    AUG 27, 2020 7:00 AM PDT
    DATE: August 27, 2020 TIME: 7:00am PT Novel anti-viral medications, including biologics and small molecule inhibitors, as well as serologic diagnostic tool sets are in urgent demand to fight...
    NOV 05, 2020 7:00 AM PST
    Add to Calendar Select one of the following: iCal Google Calendar Outlook Calendar Yahoo Calendar
    C.E. CREDITS
    NOV 05, 2020 7:00 AM PST
    Add to Calendar Select one of the following: iCal Google Calendar Outlook Calendar Yahoo Calendar
    DATE: Date needed, 2020 TIME: Time needed Exosomes are a population of naturally occurring mobile, membrane-limited, 30 – 100 nm in diameter, extracellular vesicles containing a large...
    Loading Comments...
    Show Resources