Knockout (KO) Validation

By Stewart Newlove, PhD

Antibodies are widely used in pre-clinical research to study proteins and their functions in biological pathways and diseased states. These tools, with their high specificity and sensitivity, help life science researchers to easily identify proteins of interest at various expression levels.

However, a growing number of high-impact studies and publications have shown that not all antibodies are specific; contributing to the increasing amount of irreproducible pre-clinical research ^[1,2,3,4].

The most accepted method for validating antibody specificity is through knockout validation ^[5,6,7]. In knockout validation, antibody specificity is confirmed by testing the antibody on a knockout sample or cell line, which does not express the target protein, alongside a normal (or wild-type) cell line. The target protein is not present in the knockout sample or cell line as the gene encoding the protein is inactivated (or "knocked out") through replacement or disruption of DNA. The data is compared side-by-side and if the antibody is specific there will be no detection in the knockout sample or cell line but specific detection in the normal cell line.

Antibodies.com has incorporated knockout validation, using CRISPR/Cas9, as part of its standard antibody validation process. The knockout validated seal indicates that an antibody, in addition to being validated in the recommended applications and species, has had its specificity confirmed by knockout validation.

Western blot analysis of extracts from normal (control) and EGFR knockout (KO) HeLa cells using Anti-EGFR antibody (1:500).

• Lysates: 25µg per lane.
• Secondary antibody: Goat Ant-Rabbit IgG (H+L) (HRP).
• Secondary dilution: 1:10,000.
• Blocking buffer: 3% non-fat dry milk in TBST.
• Detection: ECL Basic Kit (RM00020).
• Exposure time: 1 second.