Are your antibodies lying to you? Why antibody validation is critical 

Dr. Sean Devenish


May 30, 2024

Antibodies are proteins that bind with high affinity and specificity to target molecules. They are one of the most used tools in protein biology and are playing an ever-increasing role as therapeutics [1]. Some have been produced to directly recognize proteins of interest, while others bind short peptide chains (peptide tags) that are bound to proteins to enable detection and/or isolation.

While antibodies are tremendously powerful tools that have revolutionized biological research, the commonly held belief that they are consistently able to bind their target is an unfortunate over-simplification. In truth, antibodies are sensitive to post-translational modifications of their target and to context considerations, with residues surrounding tags able to modulate binding efficiency [2,3]. These findings highlight the critical importance of proper antibody validation for each specific application, as they may not function as intended against certain targets and contexts [4].

antibody validation

Antibody validation is commonly neglected

Pathologist David Rimm from Yale School of Medicine who was not part of the research team that published the Science Signaling studies commented:

This issue keeps popping up its head because people don’t follow directions for antibody validation. [Researchers] don’t take it seriously enough”. He further highlighted that unvalidated antibodies continue to be used in peer-reviewed publications.

The claims in the Science Signaling studies were supported by pharmacologist Thomas Wieland of Heidelberg University: “These papers, like others, clearly indicate that even commercially available antibodies need thorough evaluations for the respective purposes for which they will be used.”

Egon Ogris (Max Perutz Labs Vienna), lead author of the two studies is likewise concerned by the use of unvalidated antibodies. Work in his lab with a commonly used commercial antibody identified unexpected behavior, leading him to investigate further as a “service to the community”.

People need to know the danger attached to antibody use, and if you do not want to be tricked by your antibodies, then you need to validate them.”

― Egon Ogris, Medical University of Vienna


Context can eliminate antibody binding

Ogris’ group studies the ubiquitously expressed multi-subunit enzyme, phosphatase 2 (PP2A), which plays a role in many cellular functions and is involved in a range of diseases including neurodegeneration and cancer. To examine one of the subunits of the enzyme, Ogris et al. appended the Myc peptide tag to it, however, because they used different enzymes to add the Myc tags, they developed two variants of the tagged subunit that were identical except for a handful of amino acids at the junction between the tag and the protein [2].

When the team attempted to use 9E10, a commercially available anti-Myc antibody, to detect the two Myc-PP2A variants they found that one version of the protein gave a strong clear signal while “the other was hardly visible”.

The only difference was these four or five amino acids,” Ogris says, suggesting that these residues were the source of the variable behavior observed. To learn more, his team developed a new anti-Myc antibody, A46, and showed that this new antibody could bind to both Myc-PP2A variants equally well. Orgis’ team subsequently investigated four other commercially available Myc antibodies, and found that three of these showed differential affinity for the Myc tag depending on the surrounding amino acid context.


Post-translational modifications can prevent antibody binding

In their second study, Ogris et al. investigated the consistency of binding of antibodies targeting the catalytic subunit of PP2A [3]. There are a range of these antibodies commercially available, but it is not clear how they respond to methylation of the subunit, a post-translational modification required to activate the enzyme. Of four antibodies tested, only one efficiently detected the methylated subunit, with the remaining three antibodies all showing greatly reduced affinity for the methylated form compared to the unmodified enzyme. Poor specificity was also a problem for these three antibodies as they were able to bind, albeit weakly, phosphatase PP4.


A new method of antibody validation?

There may be a light at the end of the tunnel for those looking to validate their antibodies thanks to emerging technologies based on microfluidic systems. One example is the Fluidity One-M which measures the solution-phase binding affinity between proteins and is also able to determine the stoichiometry of complex formation. By measuring protein interactions fully in solution, the Fluidity One-M provides researchers an orthogonal and easy-to-use tool for antibody validation. There is a growing realization that commercial vendors do not carry out rigorous testing of antibodies prior to supply and it is essential for researchers to validate that antibodies are suitable and for their experiments. Internal validation is the only way that scientists can be confident of the behavior of their tool antibodies and ensure reproducible results [5–7].

For more information on how you can validate the size and binding affinity of your antibodies, click here.



  1. Yelton, D.E.; Scharff, M.D. Monoclonal Antibodies: A Powerful New Tool in Biology and Medicine. Annu. Rev. Biochem. 1981, 50, 657–680.
  2. Schüchner, S.; Behm, C.; Mudrak, I.; Ogris, E. The Myc Tag Monoclonal Antibody 9E10 Displays Highly Variable Epitope Recognition Dependent on Neighboring Sequence Context. Sci. Signal. 2020, 13, eaax9730.
  3. Frohner, I.E.; Mudrak, I.; Kronlachner, S.; Schüchner, S.; Ogris, E. Antibodies Recognizing the C Terminus of PP2A Catalytic Subunit Are Unsuitable for Evaluating PP2A Activity and Holoenzyme Composition. Sci. Signal. 2020, 13, eaax6490.
  4. Janes, K.A. Fragile Epitopes—Antibody’s Guess Is as Good as Yours. Sci. Signal. 2020, 13, eaaz8130.
  5. Bordeaux, J.; Welsh, A.; Agarwal, S.; Killiam, E.; Baquero, M.; Hanna, J.; Anagnostou, V.; Rimm, D. Antibody Validation. BioTechniques 2010, 48, 197–209.
  6. Voskuil, J. Commercial Antibodies and Their Validation. F1000Research 2014, 3, 232.
  7. Roncador, G.; Engel, P.; Maestre, L.; Anderson, A.P.; Cordell, J.L.; Cragg, M.S.; Šerbec, V.Č.; Jones, M.; Lisnic, V.J.; Kremer, L.; et al. The European Antibody Network’s Practical Guide to Finding and Validating Suitable Antibodies for Research. mAbs 2016, 8, 27–36.


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