BSA vs Milk
Which is better? BSA vs. Non-fat milk
Choosing which blocking agent to use is an important step when planning out a Western Blot.
There are two common blocking agents; Bovine Serum Albumin (BSA) or Non-Fat Milk. Both are used for different circumstances within a Western Blot therefore before picking one, it would be best to know which is best for your Western Blot.
Some of the key differences between using milk or BSA are the expense and availability. But there are likely some additional considerations to make, including what types of medium do either bind to and what proteins should or shouldn’t be used with a particular blocking buffer.
A blocking agent is a non-reacting substance used towards the end of a Western Blotting protocol to prevent non-specific binding of chosen antibodies. They typically block unimportant sites present on the transfer medium and the blocking agents do this by cancelling out regions of the Western Blot transfer membrane where there are no proteins present.
Recommended blocking agent concentrations:
When preparing a blocking agent, the concentration is generally between 1-5%, allowing changes to be made to the intensity of Western Blot blocking for a specific experiment. It’s recommended to start with a lower concentration and slowly increase it to 5%. For an intensely binding antibody, a 5% concentrated blocking agent is more than likely to help eliminate false positives.
Choosing between BSA and milk
When selecting between BSA and Milk, considering the antibodies and proteins used during the Western Blot is advised. Different antibodies have different binding strengths, and all proteins are expressed at varying levels. This is dependent on protein function and expression urgency in varying cell types within a system.
Cost of different blocking agents
Many researchers choose to use Milk over BSA as their blocking agent as it is the cheaper and easier option available. Its general application is for experiments where the antibodies have a good binding ability and the target protein is expressed at relatively high levels. When using Milk as a blocking agent, it’s not necessary to gradually increase the concentrations, starting at 5% is recommended, making it a simple and easily adaptable blocking agent for a Western Blot experiment.
Binding strength as a factor when making the decision
However, Bovine Serum Albumin (BSA) is a blocking agent tailored towards antibodies and proteins with special circumstances where the antibody used has low binding strength or the protein is expressed at low levels. This is the first indicator as to which should be chosen as a blocking agent. Protein expression levels and the binding affinity strength have to be considered when making this choice.
Number of proteins found in the blocking agents
Its also worth noting that Milk contains several of its own proteins, whereas BSA is a single purified protein. This can have significant consequences on a Western Blot data if the less appropriate blocking agent is used.
Phospho-specific antibodies and BSA
If the antibodies used during a Western Blot are phospho-specific, it is best to use BSA as the blocking agent as proteins such as Casein, which is found in milk, is a phosphoprotein and can indeed react with the phosphor-specific antibody via non-specific binding. Another implication which can come about from this is that the background could increase significantly if the washing process is not thorough. In cases like these, BSA is best.