Knockout Cell Lines from B-MoGen
5 Pillar Validation
Loading Control Handbook
KO Antibody Validation
Custom Knockout (KO) Cell Lines are available through our sister company B-MoGen Biotechnologies, Inc.
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CRISPR/Cas9 Knockout Lysates
The clustered regularly interspaced short palindromic-associated protein 9 system (CRISPR-Cas9) provides a powerful tool to specifically and efficiently introduce genomic-modifications. Specificity is encoded by a single-guide RNA (sgRNA), which targets Cas9 nuclease to genes of interest leading to double strand breaks. Subsequently, recall of DNA repair mechanisms introduce insertions and deletions (indels) which efficiently lead to bi-allelic mutations and gene silencing.1
Application of this cutting-edge CRISPR/Cas9 gene-editing technology for the generation of knockout cell lysates and cell lines ensures the development of useful tools to validate antibody specificity and elucidate gene function.
The use of genetic negative controls has been proposed by the International Working Group for Antibody Validation as one of five key criteria for ensuring antibody specificity.2 Specificity, selectivity and fidelity of antibody-target interactions often varies based on the intricacies of specific analytical applications (e.g., immunoblot vs. immunostaining). Double knockout cell-lysates (Western Blot-WB) and cell-lines (Immunocytochemistry-ICC) are the best negative controls to quickly and confidently prove an antibody’s specificity for a target of interest and ensure reproducibility.3
In WB analysis, an initial indicator of antibody specificity is the presence of a single band at the expected target size, however proper antibody validation requires the use of adequate controls.
Genetic Strategies Validation. Knockout Validated: Lactate Dehydrogenase B Antibody [NBP2-53421] - Western blot shows lysates of 293 human embryonic kidney parental cell line and LDHB knockout (KO) 293 cell line. PVDF membrane was probed with 1 ug/ml of Rabbit Anti-Human LDHB Polyclonal Antibody (Catalog # NBP2-53421) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog #HAF008). Specific band was detected for LDHB at approximately 35 kDa (as indicated) in the parental 293 cell line, but is not detectable in the knockout 293 cell line. This experiment was conducted under reducing conditions.
Because CRISPR/Cas9 gene-editing technology enables complete removal or "knock out" of both alleles of the gene encoding the target protein, antibody specificity is confirmed by demonstrating that a protein band is only present in the wildtype and not the KO cell lysate in WB analysis. To confirm differential expression, it is also important to analyze a separate target protein as a loading control (e.g., GAPDH) to demonstrate that all samples were loaded and transferred equally across wells.
Novus continues to support efforts geared at ensuring antibody specificity by adding more KO validated data to our antibody datasheets. Take your research a step further with B-MoGen's genome-wide selection of knockout cell lines.
Useful applications include:
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- Peng, J., Zhou, Y., Zhu, S., & Wei, W. (2015). High-throughput Screens in Mammalian Cells Using the CRISPR-Cas9 System. The FEBS Journal, 1–8. https://doi.org/10.1111/febs.13251
- Uhlen, M., Bandrowski, A., Carr, S., Edwards, A., Ellenberg, J., Lundberg, E., … Yamamoto, T. (2016). A proposal for validation of antibodies. Nature Methods. https://doi.org/10.1038/nmeth.3995
- Bordeaux, Jennifer et al. “Antibody Validation.” BioTechniques 48.3 (2010): 197–209. PMC. Web. 24 May 2018