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.

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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).


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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.

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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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