Several practices for antibody validation have been previously described [1-3]. These include evaluating the immunolabeling pattern of a particular antibody with positive and negative controls, assessing colocalization with orthogonal markers, and using knockout or knockdown cell lines.

We appreciate the time it takes to submit a knowledge-base entry. For this reason, we elected for a validation standard that encourages rigor without being oppressively burdensome. Furthermore, the knowledge-base is designed to be self-correcting, allowing entries to be refined as more people contribute their data:

If you have any questions, please post on the knowledge-base GitHub discussion forum. Thank you!


  1. Hickey, J. W. et al., “Spatial mapping of protein composition and tissue organization: A primer for multiplexed antibody-based imaging,” Nat Methods, vol. 19, no. 2, pp. 284–295, 2022, doi: 10.1038/s41592-021-01316-y.
  2. Uhlen, M. et al. “A proposal for validation of antibodies,” Nat Methods, vol. 13, no. 10, pp. 823-827, 2016, doi:10.1038/nmeth.3995.
  3. Bordeaux, J. et al., “Antibody validation,” Biotechniques vol. 48, no. 3, pp. 197-209, 2010, doi:10.2144/000113382.