If you work in a lab that regularly does immunoimaging, there’s likely a large collection of antibodies in your lab. Perhaps you’re even in that mythical place where antibodies are well-organized, documented, and easy to find (though we might need to see it to believe it.) As you browse the collection, you'll likely find some proteins of interest for which you have several antibodies in stock. Why are there so many options for a single target protein?
The answer is simple: epitope availability. An antibody can only bind to a target, or epitope, that it can access. If the epitope is hidden, perhaps in the pocket of a folded protein, or blocked by something bound to the protein, the antibody can't bind to it. As proteins change configurations or binding partners, the epitopes available for binding change as well. If this change hides the epitope that the antibodies used to recognize the protein, your antibody will no longer be effective. On the other hand, if it opens up an epitope that was previously hidden, an antibody might suddenly work that didn't before. This is most often an issue with monoclonal antibodies, which only recognize one epitope. (It can sometimes be an issue with polyclonal antibodies, so it's good to keep epitope availability in mind whenever you're working with antibodies.) Conformation changes usually fall into one of two categories: ones induced by the researcher and ones that happen naturally.
First, let’s think about induced conformation changes. Several types of assays, such as ELISAs and Westerns, frequently denature the proteins as part of the sample prep. This involves using heat and/or chemicals to break some of the bonds keeping the protein folded, changing it from its native state to a less folded, or denatured, one. This enables absorbing of the proteins to the microtiter walls for ELISAs and a more accurate separation of proteins by mass:charge ratio in an SDS-page gel. As the protein unfolds, new epitopes may appear, while others may get hidden. Monoclonal antibodies suitable for a Western or ELISAs may therefore often be specific to denatured versions, or even to just that particular assay.
|Fig. 1: A native protein (left) is denatured, allowing the antibody to bind to a previously hidden epitope (right). Created withBioRender.com.|
If you’re feeling relieved that you’re working with native proteins instead of denatured ones - well, don’t relax just yet. Proteins often change shapes and epitope accessibility naturally, by binding to other proteins, changing locations, or even just as part of performing a specific function. A transmembrane protein that is being used as a marker for flow cytometry, for example, can only be recognized by antibodies that bind to the epitopes that are available on the outside of the cell. The same protein may have different epitopes available if it is being selected out of a complex mix of proteins that have been extracted from a cell, instead of when it is attached to a cell membrane.
Picking the Right Antibody
It can get even more complex if you’re looking for proteins that are interacting with one another, or if you’re looking for a protein of interest while it’s in a transition state or specific configuration. So how do you ensure that you have the right antibody for the protein as it exists in your assay?
First, try to understand, to the best of your ability, what factors may be affecting the protein’s configuration in your assay. Did you have a denaturing step? Do you want to capture the protein bound to anything? Is it a transmembrane protein, or is it know to translocate or fold in certain conditions? It can help to visualize an abstract protein during your sample prep process, imagining how it starts and if any steps could affect its configuration. You don't necessarily need to know where the epitopes are, just if the available epitopes could be affected by, or critical to, the assay.
Next, look for antibodies that have been validated for the type of assay that you are performing. If they’re not available, the next best thing is a protein that has been validated for an assay that uses similar sample preparation - for instance, if you cannot find an antibody validated for Western blots, but find one validated for ELISAs, it has a good chance of also working for a Western because they both typically use denatured protein samples. If, however, you are performing an immunohistochemistry assay, you may want to rule out monoclonal antibodies that have been validated solely for denaturing assays. Many antibodies will note if they’re intended for a specific condition or configuration.
Once you’ve selected an antibody, or multiple antibodies, you’ll need to validate them for your protein and its configuration in your assay. For some assays, you may only need to validate for recognizing the protein following your sample prep steps; ie, you may only need to validate on denatured proteins or on live cells. But for others, you may need to validate that it recognizes the protein of interest in specific configurations or conditions. In those cases, pay special attention to designing your controls in validation, as you may need to test different configurations and conditions to understand exactly what your antibody recognizes.
Understanding how the available epitopes in any given protein might change depending on the conditions and the assay can help you avoid a great deal of frustration when designing and troubleshooting immunoimaging assays. It can also elucidate information about what a given protein is doing or changing in different conditions. Just remember to keep good records on which antibodies are suitable for which assays and conditions when you’re building your antibody library!
References and Resources
More resources on Addgene’s blog
- Antibodies 101: Monoclonal Antibodies
- Antibodies 101: Polyclonal Antibodies
- Antibodies 101: Introduction to Antibodies
Resources on Addgene.org
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