After a sample has been transferred from the gel to a blotting membrane, most Western blotting protocols include a blocking step before incubating the blot with the primary antibody. Understanding how blocking works is key to maximizing the quality of your Western blot, improving specificity, sensitivity, and the signal-to-noise ratio of your data.

Why is the Western blot blocking step important?

Figure 1. Choice of blocking agent can be the difference between high background (A) and excellent signal-to-noise ratios (B). 

How does Western blot blocking work?

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). The blocking buffer covers 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.

Which blocking buffer is right for my Western blot?

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. 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.

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.