Choosing the right antibody is perhaps not the first thing you think of if you experience challenges and need to start troubleshooting your immunoprecipitation (IP) experiment? The fact is, antibody selection can be make or break for the success of your IP experiment.
In my earlier post, I shared some tips for successful immunoprecipitation. In that post, I spelled out the keys to success in immunoprecipitation—strong protocol, robust capture resin, suitable format, and the right choice of antibody. Antibody selection can be a bit of an art—here is what I have learned about choosing the right antibody:
Get the right antibody—right from the start!
Check that an antibody has been tested for the IP experiment you are planning to perform. This one seems simple, but worth adding to your mental checklist. I realize this can be difficult since most companies do not provide the data required to decide how and if the antibody is validated for a specific application.
For antibodies against human antigens, the online reagents portal Antibodypedia (antibodypedia.com) has cataloged more than 1.8 million antibodies and rated the validation data available for various applications (1).
Only the best (antibodies) will do
Commercially available antibodies are frequently used for IP applications but they are common causes of problems too. Batch-to-batch variability between antibodies can produce different results between IP experiments (2). In addition, antibodies often cross-react with protein antigens that display similar epitopes, which can give false positive responses.
Take the right steps in your validation!
I would venture to say that it is most likely impossible to identify a single method for characterizing antibody performance for all different applications. The solution? Take care to carry out antibody validation in an application-specific manner. Here are a few tips on how to do this.
- Look at antibody supplier’s website; antibody vendors are encouraging users to report their own data and ranking on the vendor websites (3),
- Search in published literature.
- Test different antibodies during optimization of your IP method. Run a few assays to see which antibody performs best for your assay. This can save you a lot of time later.
Wave goodbye to high background
Polyclonal serum contains antibodies against multiple binding sites on the antigen, which helps stabilize the immunocomplex and gives you a stronger signal. The problem is, polyclonals may bind nonspecifically and can be a major source of high background levels and poor results. Why not try a monoclonal antibody instead? They bind more specifically to the antigen of interest, which reduces background.
High background may also be due to nonspecific binding of proteins to the resin. My recommendation is to include the resin you are using in your IP experiment as a control to see if the background is caused by the resin itself or something else.
Antibodies can be produced in-house or purchased commercially. In both cases, affinity chromatography can be used to remove antibodies recognizing other antigens. The antibody concentration and purity can be checked by SDS-PAGE before you start your IP experiment.
Have you experienced problems with your IP experiments you would like to share?
Check out or solutions for immunoprecipitation in the tabs below.
- Baker, M. Reproducibility crisis: blame it on the antibodies. Nature 521, 274–276 (2015).
- Moser, J. J. et al. Optimization of immunoprecipitation–western blot analysis in detecting GW182-associated components of GW/P bodies. Nat. Protoc. 4(5), 674–685 (2009).
- Uhlen, M. et al. A proposal for validation of antibodies. Nat. Methods 13, 823–827 (2016).
- nProtein A Sepharose 4 Fast Flow
- Protein A Sepharose CL-4B
- Protein G Sepharose 4 Fast Flow
- GammaBind Plus Sepharose
- Immunoprecipitation Starter pack
- Protein A HP SpinTrap
- Protein G HP SpinTrap
- Protein A HP MultiTrap
- Protein G HP MultiTrap
- Protein A Mag Sepharose
- Protein G Mag Sepharose
- Sera-Mag Speedbeads Protein A/G