Which is best? BSA vs Non-Fat Milk

So, you are about to carry out a western blot. As a researcher you may be asking yourself one of the most common, and appropriate questions there is in regards to setting up your experiment. “Which blocking buffer/blocking agent do I use?”

You’ve most likely identified a few of the key differences between using milk or BSA, such as the expense or the availability. But there are likely some additional considerations to make.

Like, what types of medium does either bind to? Or are there any proteins I really should or shouldn’t be using with my chosen blocking buffer?

We all already know that a blocking agent is a non-reacting substance used towards the end of a Western Blotting protocol to prevent non-specific binding of your 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.

Otherwise, your data may not be at its best, and you don’t want all those precious research hours to result in disappointment.

When you make up your blocking agent the concentration is generally between 1-5%, this allows you to tailor the intensity of Western Blot Blocking for your experiment.

It’s always a safer idea to start with a lower concentration and then slowly work your way up to 5% (Going over 5% means you risk blocking protein regions as well!). For an intensely binding antibody, a 5% concentrated blocking agent is more than likely to help eliminate those infuriating false positives.

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.

So which do you choose?

Selecting between BSA and Milk you must always consider the antibodies and proteins used during your Western Blot. Different antibodies have different binding strengths, and all proteins are expressed at varying levels.

This is obviously dependant on protein function, expression urgency in varying cell types within a system etc.

Many researchers choose to use Milk over BSA during 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 your blocking agent, it’s not necessary to gradually work your way up the concentrations, starting at 5% is completely fine, making it a simple and easily adaptable blocking agent for your Western Blot. However, Bovine Serum Albumin (BSA) on the other hand, 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 you should choose for your blocking agent. Is your protein expressed at high levels? How strong is the binding affinity of your antibody?

It's also worth noting that Milk contains a number of its own proteins, whereas BSA is a single purified protein. This can have significant consequences on your Western Blot data if the least appropriate blocking agent is used.

If the antibodies used during your Western Blot are phosphor-specific antibodies 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.

Conclusive Rule of Thumb

A general summary of conclusion is that there is a typical rule of thumb to follow when selecting your blocking agent. The Rule of thumb indicates the pros and cons to each of the two options for your experiment.

Non-Fat Milk

Pros

• Much cheaper
• Blocks better than BSA
• Easy preparation
• Cannot be used with antibodies which target phosphoproteins
• Cannot be used with streptavidin-biotin-based systems as milk also contains biotin
• Lowers the sensitivity of some commercially available anti-His monoclonal antibodies.

Cons

• Cannot be used with antibodies which target phosphoproteins
• Cannot be used with streptavidin-biotin-based systems as milk also contains biotin
• Lowers the sensitivity of some commercially available anti-His monoclonal antibodies.

BSA

Pros

• Provides defined results – only one protein means lower risk of cross-reactivity
• As Albumin is not generally phosphorylated, it can be used with phosphoproteins.

Cons

• More expensive
• Not recommended for lectin probing as its carbohydrates may increase background.