Your literature review is almost done, your cell or animal model is established, and your research question is solidified. Now all that’s left is designing your experiment — and ordering your supplies.
Selecting the proper antibody for your research project is a crucial step that can make or break your experience at the bench. While there is no shortage of options in the market, taking a moment to carefully consider your research goals before making your purchase can save you time and money down the road.
An antibody that works for one set of experiments may not be the best option for all applications. Therefore, it is important to examine your options each time you start a new research plan.
While most experiments will require at least some optimization, there are a few key questions to ask before making any new antibody purchase:
What is the source of my sample?
Perhaps the most fundamental consideration, the source of sample you intend to test — human, mouse, goat, rat or any other — dictates which antibodies will bind to your molecule of interest. The antibody must be produced in a different species than the source of the sample (e.g. a goat antibody may be used against a human tissue sample). Note also when making your selection that secondary antibodies must be produced by a species different from both the sample and the primary antibody.
Because of protein homology across species, some antibodies may cross react, binding to sample proteins from multiple species. It is important to note these indications in the product datasheet or on the company’s website.
What application do I intend for this antibody?
While the protein of interest in your sample may stay the same, the experiment you decide to run may change, which can make it necessary to change your antibody. Consider the sample preparation carefully: Western blot and IP protocols require proteins to be denatured, exposing antibody binding sites that may not be exposed during ELISA, IHC or FACS. Additionally, the application you choose may require specific secondary antibodies (e.g. fluorescently conjugated or enzyme linked) for proper detection and measurement. Most manufacturers indicate the intended application(s) of an antibody in the product information.
What epitope do I want to detect?
The epitope is the region of a protein or other molecule to which the antibody binds. Larger proteins have more epitopes, meaning a greater number of unique antibodies can bind to them. Denaturing a protein exposes additional epitopes that may be otherwise concealed. When designing an experiment, it is important to distinguish between monoclonal and polyclonal antibodies. Monoclonal antibodies recognize a specific epitope of a protein and are produced from a single B cell, whereas polyclonal antibodies recognize multiple epitopes and are produced by different B cells. (A polyclonal antibody is really a collection of various antibodies that each has their own specific epitope.) Each type has advantages and disadvantages, and each can prove useful in the right application.
Is the antibody validated?
Before investing time and money into a pilot experiment or full-scale study, it’s wise to look for external validation of your protocol. Many company websites will provide preliminary validation and sample results of their most popular antibodies. Given the number of products, however, it is common to find antibodies available for purchase with no previous validation. In these cases, it is a good idea to inquire about free samples, which many companies will provide in order to facilitate pilot studies and optimization experiments.
In other cases, you may elect to purchase the same antibodies used in previous experiments or antibodies cited in literature related to your research. When doing this, it is important to carefully check the product and company information, as different production batches may have different reactivity or other properties. It is also necessary to consider whether you intend to use the antibody in the same application, as discussed previously.
Is the company a reliable source?
Long-term projects can become derailed if a supplier discontinues or alters the antibody you have been using for your experiments. Consider the reliability and reputation of the company carefully before committing to their product over an extended period. Contact their customer service representatives with any questions, and use the responses to gauge the company's technical merits. This will help bring peace of mind when you experience technical hurdles later on.
Finally, compare the company’s prices to its competitors for similar products and ask if they provide discounts for bulk or repeat orders. Remember that small savings on each order can add up over the course of a multi-year project.