Knockout Validation
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What is Knock-out antibody validation?
Antibodies are a vital part of numerous pre-clinical protein-related research and development. Their sensitivity and exceptional specificity have made them an irreplaceable tool for many researchers, but numerous studies have proven that this trait is not always the case: sometimes antibodies are not created equal and possess different characteristics, giving them the potential to completely throw off an experiment or even a whole study altogether.
This uncertainty in their qualities is the reason why antibodies are validated and why different methods of validation are used. One of the best and frankly foolproof methods for validation is the “knockout” validation.
The five pillars of antibody validation:
Several years ago, in 2016, the International Working Group for Antibody Validation came up with five pillars for validation, each one being a different way of successfully tackling problems with validation.
The first pillar is represented by the Genetic Strategy Validation: where the expression of the protein of interest is compared before and after a process called knockout mainly using CRISPR/CAS9 or siRNA/shRNA.
By using CRISPR-CAS9, the DNA is cut at a specific site and the gene can be removed, added or cut where necessary. This way, this procedure can be used to create knockout cells, which do not express the targeted protein of a specific antibody. Furthermore, this procedure is widely used because of its accuracy, speed and low cost, but the biggest flaw of this method is that altering DNA can lead to cell death, which can be a major issue if the targeted protein is essential for cell death.
On the other hand, using small interfering RNA(siRNA), which is short, double-stranded pieces of RNA can cause the knockdown of a cellular mRNA sequence that we are interested in.
This type of RNA cleaves the target mRNA at a specific site and the mRNA is later broken down by the cell, resulting in a loss of protein expression, which is a short-term change compared to the CRISPR-CAS9 method. Several issues have occurred mainly with off-target effects of siRNA, but numerous studies now ensure that these concerns have been dealt with.
What is 'knockout' validation?
With all the different methods of antibody validation on the market, there will always be a margin of error about the exact specifications and uses of an antibody, this being the reason why this particular method of validation is the most accepted and used as a standard throughout the life sciences research areas.
Essentially, this process confirms the specificity of an antibody by testing it either on a cell line or on a knockout sample, on which the protein of interest is not expressed alongside a normal cell line. The target protein is missing from the cell line or knockout sample because of the gene responsible for encoding this protein not being present and “knocked out” via disruption or replacement of the DNA.
After the experiment is conducted, the results are compared and, in the event, that there is no detection in the KO sample or cell line, but specific detection present in the normal cell line, the antibody can be categorised as a specific.
Furthermore, conducting Western blot using knockout cell lysate is a pioneering method for validating antibodies. In this variation of knockout validation, the parental cell line is paired with a KO line for WB analysis.
If the antibody is exceptionally specific it will only react with the protein of interest and a right molecular weight from the WT cell lysate and not react with the knockout cell lysate.
Why bother using knockout validation?
Numerous times antibodies have proven themselves quite hard to reproduce correctly, often resulting in faulty specificity or not reacting with their targeted protein. All these issues combined with binding to the wrong proteins and cross-reactivity need an efficient and highly reliable method to sort out this so-called “reproducibility crisis”.
This is where KO validation proves itself to be a strong and reliable technique to analyse and assess different antibodies’ qualities and specificity, resulting in a much smaller margin of error when performing experiments and having trust in the quality of reactants used.
As we know, antibodies aren’t the cheapest materials and combined with the equipment needed to use them correctly the cost of a study can get quite high, which is a problem for the majority of scientists who are self-funded of are lacking enough funds to redo experiments.
Knockout validation ensures the quality and proper uses of different antibodies, so the need to buy larger quantities just in case something goes wrong, or they lack proper validation is not an issue you have to bother with.
With clear recommended species and applications, together with confirmed specificity, using the antibodies and receiving objective results is one of the most important reasons why you should consider using KO validated antibodies.