Say you're in the lab, searching for an antibody on your computer. After mining the data from the resulting loads of possibilities, your choice falls between 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 highly frustrating, especially when under pressure to publish the study. The number one reason why an antibody doesn't perform as promised is that it has not been adequately 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 unpleasant experiences, do some research before your purchase and ask yourself five straightforward yet fundamental questions:

1. Is the antibody specific to my target protein?

First, ask yourself if the antibody you want to buy has been proven specific for your target protein. A lack of antibody specificity can invalidate the results of an experiment and thereby impact scientific reproducibility. However, proving that an antibody is specific is not as difficult as you 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 that can supply the 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, another critical validation aspect is ensuring that the antibody has been tested for cross-reactivity with closely related proteins.

2. Is 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, tissue type, and target protein concentration, can impact an antibody's performance. Also, the sample treatment for different applications influences epitopes on the target protein. Every step in the application protocol, such as the fixation and embedding method, time of fixation, and the reagents' quality, can all impact your antibody's function.

Suppose your goal is to use the antibody for immunohistochemistry or immunofluorescence. In that case, you must be sure that the antibody can recognize its target when used for these applications specifically. This must be clearly stated on the antibody data sheet 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 manufacturers have not tested your application, it is your responsibility to determine whether the antibody works as needed.

 

 

3. Is the information accessible?

Time is precious in the lab, and the temptation to throw the antibody data sheet in an already full drawer is high. Don't do that. Do you have to read it? Absolutely yes! Antibody data sheets are valuable 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, and alternative antibodies with a few images to prove it.

All validation and application data must be fully comprehensive and easily accessible. For example, 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 are presented together with appropriate positive and negative controls.

Make sure that the original staining images and tissue identification, species reactivity, and purification methods are available on the vendor website.

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

4. Is reproducibility certified?

An essential 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 crucial to ensure that analyses and assays can be repeated over time and by other researchers, giving the same results. Unfortunately, lot-to-lot variation of antibodies can cause significant problems for your experiments. So how can you maintain the reproducibility of your experiments?

Look for the original manufacturer of the antibody, who has complete control over the validation and lot-to-lot reproducibility.

In addition, 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 with a strong scientific support team who would go far to look at your need and help you troubleshoot.

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 experts in the product and its methodology, you can be sure that they will assist you accordingly.

 

Remember, the first and most crucial 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 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 five essential questions, you now know what to look for when buying your next antibody, and your chances are much better that you will succeed with your experiment.