A Quick Guide to Antibody Validation

Panyue (Penny) Hao

Panyue (Penny) Hao
Jul 31, 2019 1:43:10 AM

As one of the most common reagents in biology and medical research, there are more than 350,000 commercially produced antibodies available for research and clinical applications. However, the quality of the commercially available antibodies varies from vendor to vendor. Different suppliers have different protocols for validating antibodies and some researchers might want to verify the product before using them on precious samples. Here are some of the factors to examine when it comes to antibody quality.

Target Specificity

One of the most important factors to consider when it comes to antibody validation is to make sure the antibody works against your target antigen. We have previously discussed using knockout samples to validate antibodies. Using a KO sample in your assays guarantees having a true negative control. But this also means that the assays will not test for antigen-antibody binding affinity and KO cell lines against your specific target gene may not always be readily available. Luckily, KO isn’t the only method out there.





 -  Simple procedure compared to other validation methods

 -  Can be quantitative

 -  Cannot differentiate between specific and non-specific signals


 -  Validates antibody against denatured proteins

 -  Determines specificity based on predicted molecular weight of target protein

 -  Factors such as post-translational modification and splicing may affect molecular weight

 -  Epitopes may not be available for binding in native state


 -  Validates antibody by knowing localization of target proteins

 -  Antibody binding may be inconsistent when tissues/cells are treated or fixed differently

IP and variations

 -  Able to test antibody specificity against proteins in their native state

 -  Not all antibodies are suitable for IP

Species Reactivity

Antibodies are usually raised using animals that are a different species to the host of the native protein. This is to maximize the possibility of evoking immune response and produce antibodies. However, this also means that sometimes the resulting antibodies may not work in your sample. Therefore, make sure to check the cross-reactivity of the antibody you purchased or test the antibody on a smaller sample first.


At ABclonal, we purify our antibodies using antigen affinity purification. Validation is completed using various applications and samples from different species. To find out more, visit us at ABclonal.com or contact us. Additionally, if you're curious about the various applications of antibodies in research, check out some of our other antibody-related blogs that we've curated here.


Bordeaux, J., Welsh, A. W., Agarwal, S., Killiam, E., Baquero, M. T., Hanna, J. A., ... & Rimm, D. L. (2010). Antibody validation. Biotechniques, 48(3), 197-209.

Tags: Reproducibility Crisis, Lab Life, PhD Life, Antibody Validation

Panyue (Penny) Hao

Panyue (Penny) Hao

Content creator, knowledge craver, all-around food fanatic thriving to survive in the fascinating yet uncharted world of biology.