5 tips about validation when choosing an antibody

Dozens of companies sell antibodies against your target proteins, and with so much choice available, looking for the perfect one can sometimes feel like a never-ending search. Read on. Here we share our 5 key points to ask yourself when choosing your next antibody.

Say you’re in the lab, on your computer, searching for an antibody. After mining the data from the resulting loads of possibilities, your choice falls among 2 or 3 candidates. Great. But which one is the right one for you? Is it a complicated decision, or a simple one? Finding an antibody that works for your specific application can be a difficult task.

Let’s say you made your choice and the newly purchased antibody arrives. Guess what? It doesn’t perform as advertised. This could be extremely frustrating especially when you’re under pressure to publish the study. The number one reason why an antibody doesn’t perform as promised is that it has not been properly validated, or not even tested, in the application you are using it. And now you are sitting there asking yourself: what do I do now? 


Do not play dice when looking for your next antibody

To avoid this unpleasant experience, do some research before buying your next antibody and ask yourself a very simple yet fundamental question: “Is this antibody validated in my specific application?”

1. Is the antibody specific to my target protein?

The first thing first to ask yourself is if the antibody you want to buy has been proved specific for your target protein. Lack of antibody specificity can invalidate the results of an experiment and thereby impact scientific reproducibility. To prove that an antibody is specific is not as difficult as you might think. Do you know the antigen sequence? Perfect! Then be sure that your new antibody binds specifically with that unique epitope on your target protein. 

Look for companies who can supply the exact immunogen sequence used to raise the antibody. Be aware that if the antibody shares 75% sequence homology with another protein it is predicted to cross-react. This is a serious consequence that occurs when the two antigens have similar structural regions. Thus, making sure that the antibody has been tested for cross-reactivity with closely related proteins is another critical validation aspect.

2. Was the antibody tested in my application?

A validated antibody meets two general criteria. Firstly, as mentioned above, it must bind to a specific target. Secondly, a validated antibody must work in a given application. Every detail such as the host, the type of tissue and the target protein concentration all can impact an antibody’s performance. Sample treatment for different applications influences epitopes on the target protein. Actually, every step in the application protocol such as the fixation and embedding method, time of fixation, and the quality of the reagents can all impact the function of your antibody.

If your goal is to use the antibody for immunohistochemistry or immunofluorescence, then you must be sure that the antibody is able to specifically recognize its target when used for these applications. This must be clearly stated on the antibody datasheet and supported by images. 

Hence, the evidence of several application protocols available for the antibody is a good sign of concrete validation efforts. So, avoid the temptation to use an antibody that is supposed to perform in many applications. Instead, pick the one designed and developed for your specific research setup. If your application has not been tested by manufacturers, then it is your responsibility to determine whether the antibody works as needed.

3. Is information accessible?

Time is precious in the lab and the temptation to throw the antibody datasheet in an already full drawer is high. Don’t do that. Do you have to read it? Absolutely yes! Antibody datasheets are useful sources of evidence for the applicability of an antibody in a specific setting. There you can read whether the antibody has been applied in your intended context, sample, and matrix combination. A good datasheet should always state the approved applications, product citations, antigen sequence, species information, protocol and dilution, current lot information, antibody stability, availability, alternative antibodies with a few images to prove it.

All validation and application data must be fully comprehensive and easily accessible. Search if successful uses in your specific applications are provided and if the references specified have utilized the same antibody. Always review the literature and trust an antibody cited in a publication only if validation data is presented together with appropriate positive and negative controls. Look to see if the exact immunogen sequence used to raise the antibody is available, including non-cropped images and tissue identification, species reactivity, and purification method.

 Check if other researchers have used the antibody in publications that addressed similar objectives. Although this doesn’t ensure that an antibody will perform as desired in your particular application, it provides an extra piece of evidence.

4. Is the reproducibility certified?

An important criterion for validation and standardization is antibody reproducibility. If you are planning a long-term study that requires multiple vials of the same antibody, you want to be sure that it will perform in the same way over and over. Reproducibility is always key to secure that analyses and assays can be repeated both over time and by other researchers, giving the same results. Lot-to-lot variation of antibodies can cause significant problems for your experiments. How can you maintain the reproducibility of your experiments?

Look for the original manufacturer of the antibodies, who have full control of both the validation and lot-to-lot reproducibility. In-house testing by the manufacturer enables a high level of confidence that the antibody provided will work every time for all the applications that it is recommended for.

5. What if I need support or feedback?

We all know that word-of-mouth recommendations from colleagues have a relatively high impact when making a choice, but what does the antibody manufacturer say?

Look for companies who would go as far as to look at your individual situation and help you troubleshoot by helping you choose the antibody that suits your experimental needs. 

In the antibody business, good technical support means that the company provides a dedicated Scientific Support Team that offers competent and user-friendly assistance to its customers. If the technical support team is built around individuals who are experts of the product and the methodology behind it, you can be sure that they will assist you accordingly.

Remember, the first and most important step is to collect as much information about an antibody as possible before using it. Time spent up-front in evaluating the information and resources that were made to validate your antibody can save you a lot of pain down the road.

There are of course many variables that can cause an experiment to fail, but don’t worry!

If you have answered these 5 basic questions, you know now what to look for when buying your next antibody and your chances are much better that you will succeed with your experiment.