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Affinity binders/antibodies are amongst the most commonly used tools in life science to study proteins and their functions within various biological pathways and diseases. With their specificity and sensitivity, these tools can help to easily identify the protein of interest at various expression levels. However, an increasing number of studies have shown that not all of these tools are specific, leading to a growing issue of experimental irreproducibility. This irreproducibility is leading to wasted time, resources and, unfortunately, the retraction of papers within the scientific community (Bradbury & Plückthun, 2015; Baker, 2015; Mullane et al., 2015Sneaker Women's Ecco Black Fashion Amrap Biom Band wHnqAOp).
One of the most accepted and trusted validation processes for antibody specificity is through knockout validation (Madhusoodanan, 2014; Rhodes & Trimmers, 2008). In a knockout validation, antibody specificity is confirmed by testing the antibody of interest on a knockout sample or cell line which doesn't express the target protein. In a knockout cell line, the antibody target is not present as the gene encoding the protein of interest is eliminated or "knocked out". This data is compared side-by-side against a normal cell line (or wild type) and if the antibody is truly specific, the antibody should have no detection in the knockout cell line, but instead a specific signal in the normal cell line. Knockout validations offers a true negative control to confirm the antibody specificity to the protein of interest (Bordeaux, et al., 2010).
Abcam has incorporated knockout based validation as a standard level of validation for its antibody products. We are starting at 500 antibodies a year, and we will knockout validate these antibodies as a part of our rigorous antibody validation process.
When you see the knockout validated seal, you can trust that the antibody has not only been validated in the recommended applications and species, but its specificity has been confirmed through our in-house knockout validation approach.
Abcam knockout validates its antibodies using the extensive library of knockout cell lines available from Horizon Discovery.
If you have any comments or questions, please feel free to contact us by phone or email, or by simply filling out an online inquiry form.
Bordeaux, et al., (2010). Antibody validation. Biotechniques 48(3):197-209. doi: 10.2144/000113382.