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A proposal for validation of antibodies

Nature Methods volume 13, pages 823–827 (2016)Cite this article

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We convened an ad hoc International Working Group for Antibody Validation in order to formulate the best approaches for validating antibodies used in common research applications and to provide guidelines that ensure antibody reproducibility. We recommend five conceptual 'pillars' for antibody validation to be used in an application-specific manner.

Antibodies are among the most frequently used tools in basic science research and in clinical assays. Despite their widespread use, as well as extensive and valuable discourse in the literature1,2,3,4,5,6, a comprehensive scientific framework for antibody validation across research applications is lacking. As a result, the quality and consistency of data generated through the use of antibodies vary greatly. This poses an impediment to the rigor and reproducibility that are the cornerstones of the advancement of science.

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Figure 1: Validation of antibodies in western blot applications using genetic strategies (a), orthogonal strategies (b), independent antibody strategies (c) and tagged protein expression (d).

References

  1. Bordeaux, J. et al. Biotechniques 48, 197–209 (2010).

    Article  CAS  Google Scholar 

  2. Baker, M. Nature 527, 545–551 (2015).

    Article  CAS  Google Scholar 

  3. Bandrowski, A. et al. Neuroinformatics 14, 169–182 (2016).

    Article  Google Scholar 

  4. Bourbeillon, J. et al. Nat. Biotechnol. 28, 650–653 (2010).

    Article  CAS  Google Scholar 

  5. Bradbury, A. & Plückthun, A. Nature 518, 27–29 (2015).

    Article  CAS  Google Scholar 

  6. Saper, C.B. J. Comp. Neurol. 493, 477–478 (2005).

    Article  Google Scholar 

  7. Stadler, C. et al. J. Proteomics 75, 2236–2251 (2012).

    Article  CAS  Google Scholar 

  8. Olds, W. & Li, J. F1000 Res. 5, 308 (2016).

    Article  Google Scholar 

  9. Kanchiswamy, C.N., Maffei, M., Malnoy, M., Velasco, R. & Kim, J.S. Trends Biotechnol. 34, 562–574 (2016).

    Article  CAS  Google Scholar 

  10. Carr, S.A. et al. Mol. Cell. Proteomics 13, 907–917 (2014).

    Article  CAS  Google Scholar 

  11. Abbatiello, S.E. et al. Mol. Cell. Proteomics 14, 2357–2374 (2015).

    Article  CAS  Google Scholar 

  12. Ebai, T., Kamali-Moghaddam, M. & Landegren, U. Curr. Protoc. Mol. Biol. 109, 20.10.1–20.10.25 (2015).

    Article  Google Scholar 

  13. Lundberg, M., Eriksson, A., Tran, B., Assarsson, E. & Fredriksson, S. Nucleic Acids Res. 39, e102 (2011).

    Article  CAS  Google Scholar 

  14. Juncker, D., Bergeron, S., Laforte, V. & Li, H. Curr. Opin. Chem. Biol. 18, 29–37 (2014).

    Article  CAS  Google Scholar 

  15. Mahen, R. et al. Mol. Biol. Cell 25, 3610–3618 (2014).

    Article  Google Scholar 

  16. Sander, J.D. & Joung, J.K. Nat. Biotechnol. 32, 347–355 (2014).

    Article  CAS  Google Scholar 

  17. Stadler, C. et al. Nat. Methods 10, 315–323 (2013).

    Article  CAS  Google Scholar 

  18. Marcon, E. et al. Nat. Methods 12, 725–731 (2015).

    Article  CAS  Google Scholar 

  19. Sjöberg, R. et al. N. Biotechnol. 33, 582–592 (2016).

    Article  Google Scholar 

  20. Jositsch, G. et al. Naunyn Schmiedebergs Arch. Pharmacol. 379, 389–395 (2009).

    Article  CAS  Google Scholar 

  21. Author guidelines for J. Comp. Neurol. http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1096-9861/homepage/ForAuthors.html (accessed 11 April 2016).

Download references

Acknowledgements

The authors are responsible for all content and editorial decisions and received no honoraria related to the development of this publication. Editorial assistance in the preparation of this publication was provided by Phase Five Communications, supported by Thermo Fisher Scientific, which had no other involvement in the development of this publication.

Author information

Authors and Affiliations

  1. Science for Life Laboratory, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, Sweden

    Mathias Uhlen & Emma Lundberg

  2. Center for Research in Biological Systems, University of California at San Diego, San Diego, California, USA

    Anita Bandrowski

  3. Proteomics Platform, Broad Institute of Massachusetts Institute of Technology and Harvard University, Cambridge, Massachusetts, USA

    Steven Carr

  4. Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada

    Aled Edwards

  5. Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany

    Jan Ellenberg

  6. Department of Pathology, Yale University School of Medicine, New Haven, Connecticut, USA

    David L Rimm

  7. Office of Cancer Clinical Proteomics Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA

    Henry Rodriguez & Tara Hiltke

  8. Department of Genetics, Stanford University School of Medicine, Stanford, California, USA

    Michael Snyder

  9. Biofluid Biomarker Center, Niigata University, Niigata, Japan

    Tadashi Yamamoto

Authors
  1. Mathias Uhlen
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  2. Anita Bandrowski
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  3. Steven Carr
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  4. Aled Edwards
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  5. Jan Ellenberg
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  6. Emma Lundberg
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  7. David L Rimm
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  8. Henry Rodriguez
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  9. Tara Hiltke
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  10. Michael Snyder
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  11. Tadashi Yamamoto
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Corresponding author

Correspondence to Mathias Uhlen.

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Competing interests

M.U. is cofounder of Atlas Antibodies, Affibody Medical, and Antibodypedia. S.C. is a consultant to Biogen and PTM BioLabs Inc. A.B. is the founder of SciCrunch Inc., the technology backing for the Resource Identification Initiative. E.L. acknowledges formal links to Atlas Antibodies. D.L.R. is a consultant to Amgen, Applied Cellular Diagnostics, AstraZeneca, Agendia, Bethyl Labs, Biocept, BMS, Cernostics, FivePrime, Genoptix/Novartis, Metamark Genetics, MDAgree, OptraScan, and Perkin Elmer; he has received honoraria from Genentech/Roche and Ventana; and he acknowledges research support from Cepheid, Genoptix, Gilead Sciences, Kolltan, Perkin Elmer, and Nantomics. D.L.R. holds equity in MDAgree.

Integrated supplementary information

Supplementary Figure 1 Utilization of publicly available antibodies in different applications.

The fraction of publicly available antibodies toward human protein targets is shown with data for the following applications: Western blotting (WB), immunohistochemistry (IHC), immunocytochemistry (ICC), flow cytometry (FC), immunosorbent assays (ELISA), and immunoprecipitation (IP). The numbers are based on information from users and providers reported to the resource portal Antibodypedia7.

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1 and Supplementary Tables 1–3 (PDF 443 kb)

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Uhlen, M., Bandrowski, A., Carr, S. et al. A proposal for validation of antibodies. Nat Methods 13, 823–827 (2016). https://doi.org/10.1038/nmeth.3995

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