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Knockout Cell Lines in Antibody Screening and Validation
Why is antibody validation important?
While antibodies are among most commonly used reagents in biomedical research, validating an antibody's specificity is crucial to ensuring the absence of nonspecific binding. Non-specific binding can mislead researchers with false positive results or produce sufficient background noise as to mask the on-target binding. These false experiences caused by non-specific antibodies may lead to the loss of several months of hard work and your valuable samples [1,2].
The methods to validate the specificity of antibodies have been development – first through RNAi based knockdown, and now through CRISPR/Cas9 based knockout cell lines.
Validate antibody’s specificity using RNAi knockdown
An excellent method for controlling antibody validation is to knockdown the on-target protein with RNAi and detect a concomitant reduction in binding using Western blot or ICC. But there is a limitation that the low knockdown efficiency and the off-target effects may lead to a false result.
Antibody validation using knockout cell lines generated by CRISPR/Cas9
By far knockout cell lines generated by CRISPR/Cas9 are widely used as negative controls for antibody validation. Creative Biogene is committed to providing the best Gene Knockout Stable Cell Lines available for your antibody validation. Our premade gene knockout stable cell lines are currently used by researchers to verify that their antibodies are specific to the indicated targets. In addition to being used as the negative controls, the knockout cell lines can also be used to characterize and optimize monoclonal antibodies [3].
This has several advantages over cell line panels and RNAi:
- A knockout can be validated at the genetic level, leaving no doubt about whether the protein is present in the cell. Functional protein expression can be ablated by introduction of frameshift mutations into the coding sequence, or the epitope can be excised completely using CRISPR-Cas9. In either case, a true negative control is ensured.
- Cell lines in which expression of the gene is also confirmed by mRNA can be selected for knockout, resulting in a genetically identical (isogenic) positive and negative control.
- Consequently, the observed loss of a protein from a cell sample can be attributed directly to the alterations made in the genome at the target site, ensuring extremely high confidence in antibody specificity.
References:
- Baker M. Blame it on the antibodies[J]. Nature, 2015, 521(7552): 274.
- Bradbury A, Pluckthun A. Standardize antibodies used in research: to save millions of dollars and dramatically improve reproducibility, protein-binding reagents must be defined by their sequences and produced as recombinant proteins, say Andrew Bradbury, Andreas Pluckthun and 110 co-signatories[J]. Nature, 2015, 518(7537): 27-30.
- Davies P, Hinkle K M, Sukar N N, et al. Comprehensive characterization and optimization of anti-LRRK2 (leucine-rich repeat kinase 2) monoclonal antibodies[J]. Biochemical Journal, 2013, 453(1): 101-113.