Western Blot Troubleshooting: What to do about non-specific bands

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Samples of blots with and without non-specific bands


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…


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. We recommend switching to an engineered blocking buffer, such as the Azure Chemi Blot Blocking Buffer or Azure Fluorescent Blot Blocking Buffer. These are specifically designed to enhance specific antibody-antigen interactions and reduce non-specific binding.


Low Antibody Specificity

Some primary antibodies have low-specificity for your protein of interest. To address this, 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.

Another possible reason for low antibody specificity could be that you’re using too high an antibody concentration, causing more off-target bands. To address this issue, increase the dilution of the primary antibody. You may also need to to increase the incubation time to offset any reduction in signal.


Samples of blots with and without high background signal


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. Consider diluting the primary and/or secondary antibodies, increasing the incubation time, and incubating the primary antibody step at 4°C.

Incomplete blocking can lead to high background as well. To address this issue, 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..

We hope these solutions are helpful the next time you see non-specific bands. Making a change to the procedure or switching blocking buffers can help you achieve clear and definitive results!


Where can I learn more?

Be sure to check out Azure’s Blocking Buffers:


We also offer frees sample of our Chemi or Fluorescent Blocking Buffers!


New to Western blotting? Need to troubleshoot your Western blot?​ Want to brush up on Western blotting best practices? Claim your free Western Blotting eBook!
  1. Fang, L. (2012). Antibody Purification from Western Blotting. Bio-protocol 2(6): e133

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