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Aug 15, 2012

10 Tips for Selecting The Right Antibody

Here are steps you can take to ensure finding reliable antibodies that fit new scenarios goes as smoothly as possible.

  • Click Image To Enlarge +
    Western blot films of some validations hanging up to dry. [Proteintech Group]

    Sourcing a new primary antibody can prove problematic, especially for a novel protein or application. Finding reliable antibodies that fit new scenarios usually requires a little trial and error, but there are steps one can take to ensure this transition period runs as smoothly and quickly as possible. The tips below serve as a “how-to” guide for selecting an antibody for a new protein target in your lab, but can also be used as a general antibody purchasing guide.

    1. A few places to begin your antibody search are antibody comparison sites (useful ones include Antibody Resource, Labome, and Antibodypedia) and search engines (Google Scholar has its merits). Comparison sites allow you to weigh up cost and application data of multiple vendors in one place. Search both the gene and protein names of your target to turn up as many results as possible.

    2. Look to the literature; do any publications already feature antibodies targeting your protein of interest? If so, be sure to include these in your comparison, keeping them at the top of your list. Use your judgment here, though—has the publication used the antibody in the application you intend to use it in? Is the data up to your standards? If the answer to any of these questions is no, consider your alternatives.

    3. Your application and target of choice are usually good indicators of whether you will need a polyclonal or monoclonal antibody (see Box). They will also dictate what type of immunogen your antibody should have been raised against (see below). Being clear on these points from the outset will help you refine your search with ease.

  • Box: Monoclonal versus Polyclonal Antibodies

    If you’re new to immunochemistry it may be best to read up on the advantages (and disadvantages) of both polyclonals and monoclonals. As a rough guide: polyclonals generally offer advantages in applications like IP and IHC, either where multiple epitope recognition is desirable (e.g., IP) or where proteins have undergone significant denaturing (e.g., IHC); whereas monoclonals are good for more specific work like flow cytometry (where multiple epitope-recognition by polyclonals would lead to inaccurate data).

    Another advantage monoclonal antibodies have over polyclonal antibodies is their reliable batch consistency. They also enable experiments that would be impossible with polyclonals—such as probing for PTMs at specific amino acid residues. However, polyclonals have their merits when probing for low expression proteins or in conditions where the nature of the antigen is not known, i.e., looking for a predicted homologue in your chosen species.

  • Click Image To Enlarge +
    In-house immunofluorescence staining of HepG2 cells with Proteintech’s anti-COIL (coilin) primary antibody (10967-1-AP) and a FITC-labeled secondary antibody (green). The cell nucleus has been counterstained using DAPI (blue). As expected, this antibody exhibits a punctate-staining pattern, consistent with coilin being a component of nuclear-coiled bodies. [Proteintech Group]

    4. What do you know about your protein or epitope of interest? Where is it located? Is it subject to post-translational modification (PTM)? Identify whether you want an antibody that will recognize the whole protein, a certain segment of it (e.g., N- or C-termini), or a specific peptide sequence within the protein (e.g., a phosphorylation motif). Check immunogen details to be doubly sure your antibody will tell you what you want to know.

    5. Bear in mind the species you’re working with; if an antibody was raised in the same species as your target one, it may cause problems later at the detection stage. There are ways around this if unavoidable, though, such as including normal serum from the species in which the secondary antibody is raised in your blocking buffer.

    6. Look at the validation data on each vendor’s website, and check their depth and quality. Is there simply verification of antigen recognition by ELISA testing, or are there Western blots and immunohistochemistry (IHC) data, too (as well as any other validations)? What sorts of samples were used in these tests? Make sure you’re viewing data obtained from whole cell lysates and whole tissue preparations as opposed to those prepared with purified recombinant proteins, which is usually not an accurate reflection of how the antibody will perform in the lab.

    7. The above being said, if your application is missing validation data this does not necessarily mean the antibody will be unsuccessful; you still have a few options. Does the company offer trial samples (unlikely but not unheard of) or, better, do they cover their products with a money-back guarantee? (You can try larger volumes, confident that you’re not wasting any of your grant money with the latter option.) Money-back guarantees should be of the ‘no-quibble’ kind (make sure the company you buy from doesn’t require reams of data for you to qualify) and the best ones offer you the option of your money back as opposed to credit.

    8. If unsure of your application, polyclonal antibodies raised against the full-length protein—sometimes referred to as whole-protein antibodies—will cover most of your needs in Western blot, IHC, and immunoprecipitation (IP) experiments. They are ideal in the case of the latter two as this type of antibody recognizes more of its target and three-dimensional conformations—especially handy for IP!

    9. There is something to be said for seeking out smaller vendors and original producers rather than the larger antibody ‘supermarket’. Companies who produce their own antibodies know them inside and out and can detail everything about an antibody’s production process. They can also often perform validations at your request and can send you hard copies of their original validation data. They are also more likely to offer comprehensive custom production services in the event you are unsuccessful in your search for a pre-made antibody against your chosen target.

    10. With experimental antibodies* (those about to undergo validation), contact the company to see if a trade is possible—your future validation results in exchange for a free vial. If you can give something back to the company, even if it’s negative feedback, it is seen as some compensation for the trial, and if you do get positive data, the situation is win-win.


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