PO-146 Knockout validation of antibodies to components of the NF-kB signalling pathway

Abstract

IntroductionNuclear factor-κB (NF-κB) plays a pivotal role in inflammation and innate immunity and aberrant regulation of NF-κB can influence cancer development and progression (Ref 1). As a result, a great deal of research is now focused on targeting this signalling pathway therapeutically. In order to accurately and reproducibly examine the NF-kB signalling cascade there is a need for highly specific antibodies. We, at Abcam, have developed knockout (KO)-validated antibodies targeted to distinct elements of the NF-κB signalling pathway to maximise experimental accuracy and reproducibility.Material and methodsOur KO validation program uses the extensive library of KO cell lines available from Horizon Discovery. Target genes are mutated via CRISPR-Cas9 within a haploid cell line, resulting in a frameshift and complete loss of gene expression. Target specificity is then confirmed via western blot, flow cytometry or immunocytochemistry. These KO cell lines function as true negative controls that can be used at a large scale to confirm antibody specificity to target proteins.Results and discussionsWe present antibody validation data for seven targets in the NF-κB signalling pathway. Of the 30 Abcam products tested, we demonstrate that 22 antibodies are specific. The remaining 8 have since been removed from our catalogue due to specificity concerns. We have now validated over 1000 antibodies using this KO method, including many that are relevant to immunology and immuno-oncology research.ConclusionFollowing validation with KO cell lines as a negative controls, we can recommend 22 highly specific antibodies to proteins in the NF-κB signalling pathway. KO validation is one of many strategies that we use to ensure the specificity of our antibodies. By providing robust and specific antibodies which work the first time, we hope to help researchers to improve the experimental reproducibility and accuracy of their studies.Reference1. Bastian Hoesel and Johannes A Schmid; Mol Cancer. 2013 12: 86. doi:10.1186/1476-4598-12-86