Antibody Validation

As one of the five conceptual pillars proposed for antibody validation, Genetic Validation assesses antibody specificity by comparing protein signal in cell lysates from a control cell line that expresses the wild type protein and a corresponding isogenic gene knockout cell line in which the target gene or epitope has been knocked out using genome editing methods such as CRISPR/ Cas9. Knockout cell lines thus provide a good negative control for validating antibody specificity, and such validation is suitable for western blot, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, and immunoprecipitation applications.

As a leading provider of custom CRISPR Cell Line Genome Editing services, ASC has extensive experience in cell line model generation having engineered > 500 unique cell line models in more than 100 different mammalian cell lines. We use highly optimized CRISPR designing strategies and multi-approach protocols to generate frameshift mutations or precisely deleted targeted region(s) of the gene in your cell line of interest. We can generate custom knockout cell lines that serve as ideal negative controls to evaluate epitope specificity or to rule out non-specific binding by your antibodies.

schematic-celllinemodels-antibody-validation

We also offer alternative genetic strategies for your antibody validation:

  1. Knock-in and Affinity Tag to the gene of interest (e.g. FLAG, V5, Myc): Compare binding of the antibody directed to the protein of interest with a tag-specific antibody.
  2. Overexpression of the target protein: Knock-in the gene of interest under a high expression promoter into a cell line that doesn’t express the target protein, as a positive control to evaluate antibody binding. These types of cell lines can also be generated using our proprietary TARGATT™ technology which is ideal for site-specific gene integration and highly controlled gene expression in cell lines.

Please contact us for more details on generating your knockout cell lines, affinity tagged or protein overexpression cell lines for high quality and reliable antibody validation.

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References

Technology Papers:

  • Uhlen, M., Bandrowski, A., Carr, S., Edwards, A., Ellenberg, J., Lundberg, E., ... & Yamamoto, T. (2016). A proposal for validation of antibodies. Nature methods13(10), 823-827.
  • Bordeaux, J., Welsh, A. W., Agarwal, S., Killiam, E., Baquero, M. T., Hanna, J. A., ... & Rimm, D. L. (2010). Antibody validation. Biotechniques48(3), 197.
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