Antibody Validation by Immunoprecipitation Followed by Mass Spectrometry Analysis

  • Helena Persson
  • Charlotta Preger
  • Edyta Marcon
  • Johan Lengqvist
  • Susanne Gräslund
Part of the Methods in Molecular Biology book series (MIMB, volume 1575)


We describe a mass spectrometry-based approach for validation of antibody specificity. This method allows validation of antibodies or antibody fragments, against their endogenous targets. It can assess if the antibody is able to bind to its native antigen in cell lysates among thousands of other proteins, DNA, RNA, and other cellular components. In addition, it identifies other proteins the antibody is able to immunoprecipitate allowing for the assessment of antibody specificity and selectivity. This method is easily scalable, adaptable to different cell lines and conditions and has been shown to be reproducible between multiple laboratories.

Key words

Immunoprecipitation Antibody fragments Mass spectrometry Antibody validation 



The Structural Genomics Consortium (SGC) is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada, Innovative Medicines Initiative (EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck & Co., Novartis Pharma AG, Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and the Wellcome Trust. We also gratefully acknowledge Leif Dahllund for assistance with cell culture work and Elena Ossipova for sample preparation before MS.


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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Helena Persson
    • 1
  • Charlotta Preger
    • 2
  • Edyta Marcon
    • 3
  • Johan Lengqvist
    • 4
  • Susanne Gräslund
    • 2
  1. 1.Science for Life Laboratory, Drug Discovery and Development Platform & School of BiotechnologyKTH-Royal Institute of TechnologySolnaSweden
  2. 2.Structural Genomics Consortium, Department of Biochemistry and BiophysicsKarolinska InstitutetSolnaSweden
  3. 3.Terrence Donnelly Center for Cellular & Biomolecular ResearchUniversity of TorontoTorontoCanada
  4. 4.Centre for Molecular Medicine, Rheumatology Unit, Department of Medicine, Karolinska InstitutetKarolinska University HospitalSolnaSweden

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