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.
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.
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.
Should I use milk as a blocking buffer?
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).
Figure 1. Dramatic effect of blocking buffer on background and signal-to-noise ratio. Duplicate dot blots were processed and imaged identically except for the blocking step which was either 5% nonfat dry milk in TBST (A) or Azure Protein-Free Blot Blocking Buffer (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).
Why blocking important in Western blotting?
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.
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Frequently Asked Questions
The reason for blocking the membrane before and during incubation of the primary antibody is due to the membranes being “sticky” to any protein. We “block” in order to cover up all parts of the membrane that don’t have protein on them, including any region in between lanes, etc.
When we add the antibodies, they will only bind to the protein of interest, not the blank membrane. Blocking ensures we don’t get signal from primary antibodies sticking to the membrane. We will only get signal from primary antibodies binding to their protein of interest.
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The blocking agent may be protein or non-protein based. Some of the more commonly used blocking agents include: normal serum, Bovine serum albumin, non-fat, dry milk, Polyvinylpyrrolidone, or Tween 20. Read more about blocking agents in this application note.
Most labs use PVDF or nitrocellulose membranes, but other options are available, including nylon or cellulose membranes. Nitrocellulose membranes are ready to use, easily hydrated, and readily bind with proteins out of the box. PVDF membranes are used for Western blotting because they are most robust and sensitive. Which membrane is right for your experiment?
Get a quick overview of the steps you can take to ensure your Western blots are quantitative. This free guide also includes a troubleshooting section and tear-out quantitative Western blotting checklist.