Getting Rid of the Noise: Western Blot Blocking (2024)

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Choosing the right blocking buffer will depend on several factors, including the target of your primary antibody and the detection system you are using. Some of the most commonly used blocking buffers contain protein blocking agents. Protein blocking agents can be protein mixtures, such as nonfat dry milk or serum, or single proteins such as BSA.

Besides the blocking agent, other blocking buffer components such as detergents and buffer salts can also influence interactions between the primary antibody and its target.

Nonfat dry milk is a popular blocking agent because it is inexpensive and easy to find. However, milk contains phosphoproteins that can interfere with blots using anti-phosphoprotein antibodies. Milk can also interfere with biotin-streptavidin based detection systems. In these situations, a blocking buffer containing BSA may be preferred. Or, to avoid potential cross-reactivity between antibodies and protein blocking agents, a non-protein blocking such as PVP-40 can be used.

Figure 1 shows you the choice of blocking agent can be the difference between high background (A) and excellent signal-to-noise ratios (B). Dramatic effect of using Azure Fluorescent Western Blot Blocking Buffercompared to using non-fat dry milk on background and signal-to-noise ratio.

Because the PVDF and nitrocellulose membranes typically used for Western blotting have a high binding affinity for all proteins, your primary and/or secondary antibody may bind nonspecifically to the membrane if you skip the blocking step, resulting in high background or “noisy” blots (Figure 1).

Theblocking buffercovers up or “blocks” these nonspecific protein-binding sites ensuring that most of your primary antibody binds only to the target protein (Figure 2).

The blocking agent binds to nonspecific protein-binding sites on the membrane so antibodies bind only to their target. If you choose an effective blocking buffer, nonspecific binding can be reduced without interfering with antibody-antigen binding. Now that you understand its importance, let me tell you how long to block. Maximum blocking time should not exceed 2 hours at room temperature. Any longer and proteins can be exchanged from the membrane.

Another important factor you should consider when Western blotting is the integrity of the digital imager you’re using. It is important to use a system that provides clear results. If you’re looking for consistent results, your search ends here. Request a free, virtual demo of an Azure Imaging System, and say “Hello” to beautiful Western blots.

We hope this article was helpful. If you are still having issues, use the form on this page to ask one of our scientists a question. Ready to run your next Western blot? Make sure you have everything you by checking over this checklist for a successful Western blot.