Western Blot Troubleshooting: Non-specific bands

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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.

Samples of blots with and without non-specific bands
Samples of Western blots with and without non-specific bands

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.

  • Quick Tip: Switch 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.

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.

  • QUICK TIP: 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
Samples of blots with and without high background signal


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!

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.

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

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.

Two scientists working on Azure 600
The Azure 600 uses a 9.1MP camera to provide high resolution imaging perfect for publications. Change the sample to optics distance using adjustable height shelf for enhanced detection. Zoom into the area of interest with ROI imaging to reduce background.

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.

  1. Fang, L. (2012). Antibody Purification from Western Blotting. Bio-protocol 2(6): e133

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