• Antibody Validation

Antibody Validation

Gene knockout cell lines are an ideal negative control for evaluating antibody/ epitope specificity and non-specific binding. Genetic validation of antibodies using knockout cell lines compares protein signal in cell lysates from a control cell line that expresses the wild type protein and an isogenic gene knockout cell line in which the target gene or epitope has been knocked out. As a leading provider of custom CRISPR Cell Line Genome Editing services, ASC uses highly optimized CRISPR designing strategies and multi-approach protocols to generate frameshift mutations or precisely deleted targeted region(s) of the gene in any cell line of interest. We can generate custom knockout cell lines that serve as ideal negative controls for your antibody validation assays.

Publications

Antibody validation with gene knockout cell lines:

  • 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.

Technical Details

Antibody validation using gene knockout cell lines is suitable for: western blot, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, and immunoprecipitation applications.

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.

CONTACT US

Choosing the right genome editing technology:
Applied StemCell uses two complementary genome editing technologies to generate advanced cell line and animal models very efficiently and effectively: the CRISPR/Cas9 technology and our propriety site-specific gene integration technology, TARGATT™ for large fragment (up to 20 kb) knock-in into a safe harbor locus.

Project Purpose

CRISPR/Cas9

TARGATT™

Knock-Out (KO)

Yes

 

Point Mutation

Yes

 

Conditional KO

Yes

 

Knock-In

(<200 Nucleotide ssODN Donor)

Yes

 

Knock-In Transgenes in

Safe Harbor Loci (>2kb)

Challenging

(but limitations on size)

Yes

 (up to 20kb)

Knock-In

 (Plasmid DNA)

Challenging

(but limitations on size)

Yes

 (2 steps: KI docking site; KI transgene) 

Have Questions?

An Applied StemCell technical expert is happy to help, contact us today!