Validating antibodies with knock-out technologies webinar

​​​​How can knock-out validation support the reproducibility of your experiments?

​Overview

​​Dr. Alejandra Solache, Vice President of New Product Development at Abcam, recently discussed how we validate our antibodies using knock-out (KO) technologies. Learn about the value of gene knock-out technologies in large-scale antibody screening by watching the video below.     

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​You can see Dr. Alejandra Solache present more on antibody KO validation in this webinar from 21:03, which was a part of the “Antibody validation webinar series” hosted by the Antibody Society. For more webinars on Antibody Validation from experts in the field, you can also check out the Antibody Society's learning center.


Why use knock-out validation

Several studies have demonstrated the irreproducibility of life sciences reagents, which in the case of antibodies, frequently results from a lack of specificity to their targets of interest. This lack of specificity, related to issues such as cross-reactivity and off-target protein binding, has contributed to the so-called ‘reproducibility crisis’1,2.

KO validation is one of the most accepted and trusted validation processes for antibody specificity3,4. This robust technique can confirm the specificity of an antibody by testing it in a KO cell line, cell lysate, or tissue that does not express the target protein. A specific antibody should, therefore, produce no signal in the KO cell line but give a specific signal in the wild type sample. In this way, KO validation serves as a true negative control5.

Find out more about knock-out validation

Abcam’s knock-out validation initiative

To ensure that our antibodies are specific and provide reproducible results, we apply KO validation at a large scale. We validate our antibodies in multiple applications, including flow cytometry, western blot, and immunocytochemistry, using multiple antibodies per target. Antibodies that demonstrate specificity are given a special “KO validated” designation, whereas those exhibiting off-target binding are removed from our catalog. We have now confirmed the specificity of more than 2500 antibodies with KO validation for over 2000 protein targets.

Key learnings from Abcam’s KO validation initiative:

  • Recombinant monoclonal antibodies, in general, demonstrate superior specificity and consistency compared to traditional monoclonal and polyclonal antibodies.
  • A significant number of top-cited antibody clones have been observed to exhibit off-target effects.
  • We recommend that scientists choose antibodies that have been validated in multiple applications, ideally with the use of KO technologies. Alternatively, you can validate an antibody yourself, using the appropriate KO cell line, KO cell lysate, or tissue.
  • Confirming the specificity of an antibody can take time and effort, including the generation of a KO cell line using gene-editing technologies (on average 6 months to generate KOs). To help you accelerate your research and avoid tedious CRISPR engineering experiments, we now provide one of the largest collections of CRISPR-Cas9 KO lysates and KO cell lines.
  • All our KO cell lines and lysates are reliable, ready-to-use, and allow you to confidently progress target identification and validation without the need to first establish a KO cell line. All our KO cell lines are validated by Sanger sequencing, and many have additional western blot data to confirm KO at the proteomic level.

About Dr. Alejandra Solache

Dr. Alejandra Solache joined Abcam in December 2013 and manages Abcam’s Global New Product Development and validation teams in Cambridge, UK, Hangzhou, China as well as Eugene and Branford, CT in the USA.  Alejandra has also been instrumental in developing Abcam’s innovation strategy and product pipeline. 

Before joining Abcam, Alejandra led the Antibody and Assay Development teams as R&D Director at EMD-Millipore, where she held various positions for eight years. She trained as a life science researcher where she gained expertise in immunology, cell signaling, and epigenetics through post-doctoral fellowships at the University of California, San Francisco, and the Trudeau Institute, Saranac Lake, NY. 

Why use knockout validation?
Abcam’s knockout validation initiative
About Dr Alejandra Solache