• Antibody Validation

Antibody Validation

The Ultimate Negative Controls for Genetic Validation of Your Antibodies! Evaluate antibody/ epitope specificity and non-specific binding in gene knockout cell lines. 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, to serve as the ideal negative controls for your antibody validation assays:

  • Preliminary expression level confirmation by RT-PCR or Western Blotting
  • Gene targeting and knock-out cell line engineering using CRISPR/Cas9
  • Optional KO validation by locus-specific sequencing, RT-PCR, or western
  • Downstream integration of KO cell lines into FFPE Blocks
Application Notes

Knockout Cell Lines and Negative Control for Antibody Screening

appnote-celllinemodels-antibodyscreening

Figure. Antibody (Ab1) against a gene of interest (GOI) was validated in cell lines in which the gene was knocked out using CRISPR/Cas9.  Western blots using Ab1 do not show any binding in protein extracts from clones 1 and 2 of the knockout cell line (-/-); β-actin was used as a positive control for the assay. WT: wild type.


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!