We’ve all been there… Non-specific bands are a perennially frustrating problem in Western blotting. Here are some of the different reasons you might be getting non-specific bands and tips on how to make these unwanted additions to your Western blot disappear. If non-specific bands are giving you trouble, the issue could lie in incomplete blocking, low antibody specificity, or high background. Keep reading to see which issue you may be facing.
Issue: Incomplete Blocking
One of the most common causes of non-specific bands is incomplete blocking. Blocking buffers are used to prevent primary and secondary antibodies from binding to the membrane, or anything other than the protein of interest.
Some blocking buffers mask epitopes on your target, which decreases the binding of the primary antibody. This can make your target protein difficult to detect without long exposure times and, thus, reducing signal-to-noise. In addition, general blocking buffers such as milk or BSA are not designed to prevent non-specific binding of primary antibodies to other lysate proteins.
Issue: Low Antibody Specificity
Some primary antibodies have low-specificity for your protein of interest. A possible reason for low antibody specificity could be that you’re using too high an antibody concentration, which causes more off-target bands.
Perform the primary antibody incubation step at 4°C to help decrease non-specific binding of your antibody. Running additional purification steps on your primary antibody or generating new antibody can also help. It may also be helpful to use a wide comb so there is room to add more of your protein¹.
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Issue: High Background
Non-specific bands aren’t the only issue related to blocking. Low antibody specificity can lead to a high background on a fluorescent or chemiluminescent western blot. When the concentration of primary antibody is too high, it can bind to the membrane, causing a background signal.
Incomplete blocking can lead to high background as well. To address incomplete blocking, replace the milk with an engineered blocking buffer. Be sure to check out the Azure Blocking Buffers, including buffers for chemiluminescent and fluorescent Western blotting. We offer a protein-free blocking buffer for antibodies with high cross-reactivity to protein-based blockers as well.
If you’re looking for clear and consistent results, your search ends here. Request a free demo of an Azure Imaging System, and say “Hello” to beautiful Western blots.
Making a change to the procedure or switching blocking buffers can help you achieve clear and definitive results. We hope these solutions are helpful the next time you see non-specific bands. If you still have questions, use the form on this page to ask one of our Western blotting experts.
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- Fang, L. (2012). Antibody Purification from Western Blotting. Bio-protocol 2(6): e133
