Antibody Validation by Immunoprecipitation Followed by Mass Spectrometry Analysis

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

Abstract

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 

Notes

Acknowledgments

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.

References

  1. 1.
    Marcon E, Jain H, Bhattacharya A, Guo H, Phanse S, Pu S et al (2015) Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation. Nat Methods 12:725–731CrossRefPubMedGoogle Scholar
  2. 2.
    Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D et al (2003) Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13:2498–2504CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Marcon E, Ni Z, Pu S, Turinsky AL, Trimble SS, Olsen JB et al (2014) Human-chromatin-related protein interactions identify a demethylase complex required for chromosome segregation. Cell Rep 8:297–310CrossRefPubMedGoogle Scholar
  4. 4.
    Guruharsha KG, Rual JF, Zhai B, Mintseris J, Vaidya P, Vaidya N et al (2011) A protein complex network of Drosophila melanogaster. Cell 147:690–703CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Cox J, Mann M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26:1367–1372CrossRefPubMedGoogle Scholar
  6. 6.
    Zybailov BL, Florens L, Washburn MP (2007) Quantitative shotgun proteomics using a protease with broad specificity and normalized spectral abundance factors. Mol Biosyst 3:354–360CrossRefPubMedGoogle Scholar
  7. 7.
    Mellacheruvu D, Wright Z, Couzens AL, Lambert JP, St-Denis NA, Li T et al (2013) The CRAPome: a contaminant repository for affinity purification-mass spectrometry data. Nat Methods 10:730–736CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Dou Y, Milne TA, Ruthenburg AJ, Lee S, Lee JW, Verdine GL et al (2006) Regulation of MLL1 H3K4 methyltransferase activity by its core components. Nat Struct Mol Biol 13:713–719CrossRefPubMedGoogle Scholar
  9. 9.
    Thomas P, Smart TG (2005) HEK293 cell line: a vehicle for the expression of recombinant proteins. J Pharmacol Toxicol Methods 51:187–200CrossRefPubMedGoogle Scholar

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

Personalised recommendations