Knock-out (KO) validation: confirming antibody specificity

To ensure you have highly specific antibodies, we use KO-validation via CRISPR-Cas9 genome-editing, to deliver the reliable results that your research demands. 

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Contents

• Knock-out validation: the need and solution
• Knock-out validation: testing results
• Knock-out validated antibodies promise 
• Knock-out cell lines and lysates 

The need

Antibodies are amongst the most commonly used tools in life science and clinical research for the study of proteins and their functions within various biological pathways and diseases.

A good antibody exhibits target specificity and sensitivity, allowing it to identify the protein of interest even at low expression levels. However, an increasing number of studies have shown that not all antibodies are specific in this way, with many showing cross-reactivities with off-target proteins. This has led to the recognized issue of experimental irreproducibility.¹ These non-specific antibodies result in wasted resources and compromise the advancement of science.^2,3

The solution: knock-out validation

Knock-out (KO) validation is a robust technique used to confirm antibody specificity by testing the antibody of interest in a KO cell line or tissue that does not express the target protein.⁴ A specific antibody should yield no signal when tested in a KO cell line while giving the specific target protein signal in the wild-type (WT) cell line. In this way, KO validation serves as a true negative control to confirm antibody specificity to the protein of interest.^4,5

At Abcam, we use an extensive library of human KO haploid cell lines to perform antibody validation. These KO cell lines are generated via CRISPR-Cas9 and certified via Sanger sequencing. This type of KO cell line provides a complete loss-of-function phenotype from a single allele KO and eliminates any masking of the knockout from a second allele seen in diploid cell models.^6-8 

Knock-out validation testing results

When KO-validating our antibodies, we primarily use western blot to assess the results. A range of western blot outcomes can result from KO validation, and the following examples demonstrate how we deal with each situation at Abcam.

Specific antibody

• Result: The expected molecular weight (MW) band disappears in the KO sample.
• Next steps: KO-based specific data is added to the datasheet. The antibody is guaranteed for specificity via KO-validation. 

Partially specific antibody – ​extra bands

• Result: The expected MW band disappears in the KO sample, but extra bands are present at a distinct MW.
• Next steps: We add KO-based specificity data to the datasheet. Additional research and testing will be performed to identify the extra bands.

Non-specific antibody – ​weak signal in the KO sample

• Result: The expected MW band still present in KO at a lower intensity compared to WT. A benchmark antibody resolves a target band in WT and no band in KO. 
• Next steps: We add KO-based specific data to the datasheet. Additional research and testing will be performed to identify the reasons behind the signal in the KO sample.

Non-specific antibody – signal in the KO sample

• Result: expected MW band still present in KO at the same intensity as WT. A bench-mark antibody resolves a target band in WT and no band in KO.
• Next steps: we remove the antibody from the catalog, and recommend an alternative antibody with confirmed target specificity.

Non-specific antibody – no target band in both KO and WT samples

• Result: The antibody does not recognize the target band in both the WT and KO samples. A bench-mark antibody resolves a target band in WT and no band in KO. 
• Next steps: We remove the antibody from the catalog, and recommend an alternative antibody with confirmed specificity.

Knock-out validated antibodies: our promise

When you see the KO validated seal, you can trust that the antibody has not only been validated in the recommended applications and species, but its specificity has been confirmed through our in-house KO validation approach.

See our complete list of KO-validated antibodies.

Knock-out cell lines and lysates: edited and ready for you

The application of CRISPR-Cas9 technology has allowed us to develop an extensive range of KO cell lines and lysates, so that we can provide you with reliable antibodies, saving you time and money. Each KO cell line is individually cloned, validated by Sanger sequencing, and supplied with the parental WT control lysate to allow the biological impact of the knock-out to be assessed within a consistent cellular background.

​See our complete list of knockout cell lysates

See our complete list of knockout cell lines

References

1. Bradbury A and Pluckthun A. Reproducibility: Standardize antibodies used in research. Nature. 518:27-29 (2015).
2. Baker M. Reproducibility crisis: Blame it on the antibodies. Nature. 521:274-276 (2015).
3. Mullane K, Enna SJ, Piette J, Williams M. Guidelines for manuscript submission in the peer-reviewed pharmacological literature. Biochem Pharmacol. 97(3):225-35 (2015).
4. Zhong Z, Sassi M, Heaton S, Koch S, De Block G, Conlon D, Lochead J, Dreja H, Munro M, Solache A, Hamilton B. Large-scale use of knockout validation to confirm antibody specificity. J Immunol 200 (1 Supplement) 120.18 (2018).
5. Bordeaux J, Welsh AW, Agarwal S, Killiam E, Baquero MT, Hanna JA, Anagnostou VK, Rimm DL. Antibody Validation. Biotechniques. 48(3):197–209 (2010).
6. Bürckstümmer T, Banning C, Hainzl P, Schobesberger R, Kerzendorfer C, et al. A reversible gene trap collection empowers haploid genetics in human cells. Nat Methods. 10(10):965-71 (2013).
7. Reiling JH, Clish CB, Carette JE, Varadarajan M, Brummelkamp TR, Sabatini DM. A haploid genetic screen identifies the major facilitator domain containing 2A (MFSD2A) transporter as a key mediator in the response to tunicamycin. Proc Natl Acad Sci U S A. 108(29):11756-65 (2011).
8. Carette JE, Guimaraes CP, Varadarajan M, Park AS, Wuethrich I, et al. Haploid genetic screens in human cells identify host factors used by pathogens. Science. 326(5957):1231-5 (2009).