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.