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
Nguồn: Saint John's lab at https://stjohnslabs.com/blog/choosing-bsa-vs-non-fat-milk
