Enhanced Validation of primary antibodies: what it is and how it helps your research get published

Enhanced validation of primary antibodies. What is it? Read on and give us the chance to explain to you what Enhanced Validation is and why it makes our antibodies stand out from the crowd.

Imagine buying a new antibody from a company for the very first time. You know nothing about the brand, the quality of the product, or the dedication to customer satisfaction.

Like most researchers, you'd search for information on the vendor's website, reviews from past customers, looking for any proof that the antibody will meet your needs.

Don't waste too much time looking around. Come prepared! Do you know what to expect from a good antibody? And how and when does it meet your expectations?


Antibody validation

Validation is the process that ensures that the end product's expectations are achieved. Hence, antibody validation is the experimental proof and documentation that the specific antibody is suitable for your intended application or purpose.

Only by using an antibody that underwent a strict validation process, you are assured that it will consistently produce the expected results.

However, a strict validation means a rigorous application of different methods, and not least hard work that is usually not possible in your packed daily lab routine.

Luckily for you, thanks to recent scientific and technological progress that allows a more precise evaluation of antibody performance, some of today's commercial antibodies have superior quality. This is something you should be looking to exploit.

We are Atlas Antibodies, and we work to provide high-quality antibodies to help you generate excellent data and publish it confidently. All our antibodies are rigorously validated for specificity and reproducibility and are characterized in several applications. 

Continue reading to learn more about us and how we can provide antibodies with superior validation. 


A successful spin-off with a clear mission

Atlas Antibodies has an extraordinary story. The company was founded in 2006 by researchers from the Human Protein Atlas project (HPA). HPA is a special project aimed at mapping all human proteins in cells, tissues, and organs.

It now relies on an advanced high-tech approach that integrates cutting-edge technologies, such as antibody-based imaging, mass spectrometry-based proteomics, transcriptomics, and systems biology.

Critical for the project's success was the availability of highly specific antibodies for all human proteins. In addition, to ensure high-quality standards, the researchers from the Human Protein Atlas established a scrupulous in-house manufacturing process.

Being confident and proud of the high quality of the antibodies, they decided to make them available to the scientific community worldwide. The Triple A Polyclonals are exclusively manufactured by Atlas Antibodies in Sweden and distributed worldwide.

Today, after the eighteenth major release of The Human Protein Atlas, we have secured comprehensive proteome coverage with more than 21,000 validated antibodies, targeting proteins from ~15,000 human genes, corresponding to ~75% of the human protein-coding genes.

Our antibodies are validated at least in one of the following applications: immunohistochemistry (17500 antibodies), western blot (8450), immunocytochemistry-immunofluorescence (11000). In addition, our in-house production and validation certify that the antibodies we offer meet the highest levels of specificity and selectivity.

But wait, that's not all!

At Atlas Antibodies, we are a step ahead for systematic validation of antibodies. You should know that we apply' Enhanced Validation' in addition to our extensive antibody characterization. So read on and give us the chance to explain to you what Enhanced Validation is and why it makes our antibodies stand out from the crowd.


What is enhanced validation?

The enhanced validation is built around the recommendations promoted by the International Working Group for Antibody Validation (IWGAV) and published in Nature Methods1. This publication is an essential first step towards developing widely accepted standards for validating antibodies and ensuring consistent and high-quality antibodies for biomedical research.

The IWGAV identified five strategic "conceptual pillars" to guide antibody validation in specific research applications: 1) genetic strategy, 2) orthogonal strategy, 3) independent antibody strategy, 4) expression of tagged proteins strategy, and, 5) immunocapture followed by mass spectrometry strategy.

At least one of these validation methods (in addition to our standard validation) must be applied to each antibody to receive our Enhanced Validation stamp in a specific application. The purpose of including multiple validation methods is to increase antibody security at all levels and across numerous applications and tissues. Each pillar employs a distinct technique, offering multiple ways to achieve validation. Thus, the individual strengths of each validation method cover any gaps that other methods may lack.

Overall, by following the IWGAV guidelines, our strategy is designed to provide a scientific foundation for antibody validation and reproducibility standards.

Enhanced Validation: 5 layers of security

In this video, we explain the five methods used for enhanced validation. 



1. Genetic validation method

This strategy is based on the knock-down of the target protein using genetic methods, such as siRNA, in a suitable cell line. The staining of the antibody is evaluated by the antibody-based method through analyses of samples from cell lysates before and after knock-down of the corresponding target gene. The antibody is validated when the protein levels correspond to the decreased RNA levels.

2. Recombinant expression validation method

This strategy is based on the over-expression of the target protein in a cell line, preferably not expressing the target protein. The staining of the antibody is evaluated by the antibody-based method through analyses of samples from cell lysates with and without overexpression of the target protein. The antibody is validated by comparing the signal from the over-expressed version with the unmodified endogenous target protein. 

3. Independent antibody validation method

This strategy compares the staining pattern using two independent antibodies with non-overlapping epitopes. The staining of the two antibodies is compared through the analyses of several samples, preferably expressing the target protein at different levels. If the two antibodies generate a similar staining pattern when compared in a set of relevant samples, the antibodies validate each other.

4. Orthogonal validation method

This strategy is based on comparing the protein analysis achieved by the antibody-based method with levels of the target protein detected by a non-antibody-based method. We compare the protein levels determined by the antibody with the corresponding RNA levels. At least two samples must be used, and they should be selected so that the target protein is expressed at different levels. The antibody is validated when the signal obtained from the different samples and determined by the two independent methods correlate.

5. Migration capture MS validation method

This strategy validates the antibody with mass spectrometry (MS) analysis. Each gel lane is fractionated according to molecular weight and the fractions are separately analyzed by MS to identify how each protein has migrated on the gel. Antibody specificity is confirmed when the size detected by the antibody is equivalent to the size of the corresponding target protein detected in migration capture MS. 

Figure 1. This table lists the five different enhanced validation methods.  

We believe in open access.

To promote transparency, the details of these methods and all antibody characterization data achieved using different applications are freely accessible on our website (atlasantibodies.com). Additional data is supplied on the Human Protein Atlas portal.

Enhanced validation of an antibody includes primary data, preferably images, comparing side-by-side detection of the target in biological samples that confirm specificity towards the target, absence of cross-reactivity, and lot to lot reproducibility. For immunohistochemistry, each antibody is supplied with 500 images from 44 normal and 20 cancer tissues. 


The best in class

We build trust in our antibodies through proof and content.

As the original manufacturer at Atlas Antibodies, we have complete control over our antibodies.

You can trust that our antibodies are specific and perform consistently in your application each time you use them.

You can now rest easy knowing your research is in excellent hands. But don't just read about it. It is time for action! Are you ready to order your next antibody?


  • Uhlén, M. et al. A proposal for validation of antibodies. Nature Methods 13, 823–827, 2016