When selecting a blocking buffer for Western blotting, consider the following factors:

  • Using a blocking buffer from the same species is important. The objective is to help prevent the primary antibody from binding to the blocking agent instead of the protein of interest.
  • The detection method being used should be compatible with the blocking buffer. Problems can arise if blocking buffer interferes with the detection method. If you are unsure about compatibility a preliminary test should be done.
  • The protein of interest. Some proteins are more prone to nonspecific binding than others. If you are working with a protein that is known to be prone to nonspecific binding, you may need to use a more stringent blocking agent.
  • If you are working with Phospho proteins some blocking buffers contain proteins that are also phosphorylated, such as casein in milk. When these proteins bind to the phospho-specific antibody, they can give a false positive signal. BSA and synthetic blocking buffers should be considered.
  • Use fresh blocking buffer this reduces the chance of contaminants that can contribute to background.
  • Use the correct concentration.
  • Proper incubation times are important. The blocking steps can be automated with the BlotCycler along with incubation with primary/secondary antibody followed by multiple washing steps. This will reduce error and improve blot to blot consistency.

To optimize your experiment, we recommend trying different blocking buffers and seeing which one gives you the best results; different blocking buffers for different proteins or different detection methods.

The following are commonly used blocking buffers:

  • Bovine serum albumin (BSA). BSA is a good all-purpose blocking agent. It is compatible with most detection methods and does not interfere with the binding of most antibodies.
  • Nonfat dry milk.
  •  Gelatin works well for proteins that are susceptible to nonspecific binding and works well for chemiluminescent detection methods.
  • Synthetic blocking agents can be a good option if you are having trouble with nonspecific binding; they typically consist of small, non-immunogenic molecules that are less likely to bind to the membrane or the primary antibody.