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primary antibody

 

One of the most frustrating things in the lab is spending good money (well, ok, your Principal Investigator’s money) on an antibody only to find out it doesn’t work well in your application. You can then either spend weeks trying to make it work, abandon your work altogether or humbly ‘fess up that you made a bad purchase and beg for a second chance.

In an effort to limit your groveling, we have made a list of tips for choosing a good primary antibody.

Dare to compare

Take the time to comparison shop prior to buying an antibody. Use an antibody comparison site such as Biocompare, Antibodypedia, CiteAb, or Antibody Resource. Carefully compare names or antibody clones (the same antibody can be marketed by different companies), concentrations, price, aliquot size and recommended dilutions.

Search the literature

If you find an antibody using a comparison site, search PubMed for articles in which people have used the antibody. Read the methods and look at the figures to see if the antibody worked well. If you can’t find an antibody using one of the comparison sites, search the literature for antibodies that may not be commercially available.

Pay attention to application

The best case scenario is to find an antibody that has already been used in the exact manner in which you intend to use it. Most of us are rarely this lucky!

Search the literature, product reviews and product data sheets to determine how the antibody has been used in the past. Be mindful that antibodies that work well in one application may not be suited for another. For example, an antibody that works well in a Western may not work well in immunoprecipitations due to conformational changes in the protein. Alternatively, posttranslational modifications, such as phosphorylation, might mask or reveal epitopes recognized by antibodies (particularly monoclonal antibodies).

If you can’t find an antibody that has been used in your application, choose one that has been used in a compatible manner. For example, antibodies used in FACS analysis on live cells might also work in native immunoprecipitations which also maintain the integrity of the protein.

Additionally, pay attention to the species in which the antibody was generated and make sure it is compatible with your application. If you are going to be using mutliple antibodies at the same time (e.g. in multiplex Westerns) make sure your antibodies won’t be cross-reactive.

Species, species, species

One factor that should be taken into consideration early in your antibody search is the species of animal in which the primary antibody was generated and whether it will be compatible with your application. If you need to use a conjugated secondary antibody to detect the primary antibody (e.g. when performing immunohistochemistry) the primary antibody should be from a species as phylogenetically distinct as possible from the species of the sample. This will prevent cross-reactivity between the secondary antibody and endogenous immunoglobulins in the sample.

Check how the antibody was validated

A lot of antibodies are validated using recombinant proteins that are abundantly expressed. Or they are validated with proteins that were produced in non-native hosts such as bacteria or yeast. While these validations show that the antibody can work, they do not necessarily show that the antibody will work under alternative conditions. If you need to use an antibody to detect a rare, highly glycosylated protein in mammalian cells, then an antibody that has only been shown to detect recombinant protein in bacteria might not be the choice for you.

The type of antibody matters

Is it better to us a monoclonal or a polyclonal antibody? For some proteins, you don’t have a say in the matter – you have to go with whatever antibody is available. However, if you do have a choice, choosing the right kind of antibody might increase your success. Polyclonal antibodies, which recognize multiple epitopes, are often better in Western blots, immunohistochemistry and immunoprecipitations. They can also be more robust and detect less abundant proteins.

Monoclonals are useful in detecting conformation-specific epitopes and are invaluable when using multiple antibodies simultaneously (such as for detecting two proteins simultaneously in FACS). Due to their specificity, monoclonals often give less background. Because they only recognize a single epitope though, monoclonal antibodies can be more limited in application. But don’t let that scare you off! I have used many monoclonal anbtibodies that worked well in Westerns.

Ask about trial samples or guarantees

If you are not sure an antibody will work in your application, don’t be afraid to ask for a trial sample. Some companies will send you a small aliquot for free or charge you a small fee for a “one-time use” aliquot. You can also suggest providing them with validation data in exchange for antibody.

Some companies will guarantee that their antibody will work in a particular application. Limited risk can be very advantageous to you, but be careful of the fine print! You might only get a credit towards another antibody, or you might be asked to perform several experiments to prove it doesn’t work!

Choosing a new primary antibody can be an onerous task, but with some careful research and a little bit of leg work you can increase your chances of finding the one that works for you!

 

Photo courtesy of GoonSquadSarah.

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