Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A proposal for validation of antibodies

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

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

Authors

Corresponding author

Correspondence to Mathias Uhlen.

Ethics declarations

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)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.3995

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing