Question:
Which Blocking Agent should I use to block non-specific binding during Western blotting?
The Protein Man Says:
Western blotting is an important technique that is routinely used in research and diagnostic laboratories. Western blotting is combined with polyacrylamide gel electrophoresis, which separates proteins based on their molecular weight. Western blotting consists of the transfer of the separated proteins onto a membrane where they can be identified with specific antibodies. Western blotting is named after a similar technique, Southern blotting, which is the transfer of DNA to a membrane; a technique invented by the British biologist Edwin Southern. Northern blotting is a similar technique, but for RNA.
The key feature of Western blotting is the use of immunodetection to identify a specific protein, for example a protein marker for a disease. Once the proteins are immobilized on a protein binding membrane, usually nitrocellulose or PVDF (polyvinylidene fluoride), they can be probed with a primary antibody, an antibody specific for the protein of interest. Once bound the antibody is visualized, either with a specific tag coupled to the primary antibody or with a secondary antibody. The secondary antibody is a general antibody that recognizes the constant domain of immunoglobulin G and is species specific. So, if the primary antibody is a mouse antibody, the secondary antibody used will recognize all mouse antibodies. If a secondary antibody is used then this will carry the tag that allows visualization of the protein (see figure).
The most common tags used in Western blot are enzymes that catalyze a substrate to produce either light that is detected with radiography film, or color that is visualized on the membrane. The enzymes of choice are horseradish peroxidase (HRP) and alkaline phosphatase (AP).
Blocking Step
An additional step is crucial to Western blot and this is known as the blocking step. The blocking step is used to increase the specificity of the Western blot technique by preventing non-specific interactions. If the membranes are not blocked then the antibodies can stick to non-specific proteins due to their charge. To prevent this, the membrane is placed in a protein mixture and the proteins block the charges that would attract the antibodies. Several blocking agents are used, including dried milk powder, bovine serum albumin and casein, however modern blocking agents use synthetic and/or non-animal proteins to prevent any cross reaction with the animal antibodies.
Animal Serum Proteins (BSA)
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Commonly used due to being an inexpensive and effective blocking agent
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Use at 0.2-5% w/v concentration
Disadvantages
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High cross-reactivity, particularly with phosphor-tyrosine antibodies and biotin.
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Animal proteins can cross react with antibodies raised in animal
Non Fat Milk
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Routinely used as it is also an inexpensive and effective blocking agent
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Purified Casein often used to remove cross reacting components in milk
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Use non fat milk at 3-5% w/v, use casein at 1% w/v
Disadvantages -
High cross-reactivity, particularly with phosphoprotein antibodies
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Animal proteins can cross react with antibodies raised in animal
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Non mammalian blocking agent
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Do not cross react with antibodies raised in animals
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Clearer backgrounds
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Use at 2-10% w/v
Disadvantages
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More expensive
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Can mask some antigens and decrease sensitivity
Commercial non animal protein blocking agents and protein free blocking agents are also available that reduce that amount of cross-reactivity to animal proteins. Download G-Biosciences Western Blotting Handbook for a review of our blocking agents.
