what is better BSA or Skim Milk for PVDF membrane blocking in western blots?
Hello Navneendra Singh
The blocking step is required in Western Blot to reduce the amount of background due to non-specific bonding. You can do it either with BSA or skim milk. Now, BSA is only made up of one protein i.e., BSA at 66 kDa, whereas milk is made up of many proteins, comprising of various sizes. So, you will get a much better chance to reduce more of the background banding using skim milk. Also, many researchers choose to use skim milk over BSA as their blocking agent because skim milk is the cheaper and easier option.
Therefore, use of 5% skim milk in TBST, shaken for one hour at room temperature may be used as the blocking step in Western Blot.
However, it would be better to determine which agent is more suitable for a particular assay. To this end, antibodies and proteins to-be-detected in Western Blot should be considered. For instance, if the antibodies used during Western Blot are phospho-specific antibodies, then in that case, it is better that you use BSA as the blocking agent because proteins such as casein found in milk, which is a phosphoprotein can react with the phospho-specific antibody resulting in non-specific binding.
Best.
Non-fat milk is a very robust blocker for WB. But in some cases, it may block too heavily and you'll lose the entire band. It depends a lot on your protein of interest as well as the particular antibody. Ideally, you would optimize your western by trying a range of blocking with your antibody. For example:
1. 1% BSA
2. 5% BSA
3. 1%NFM
4. 5% NFM
Then expose your membranes side by side on one film for 1 second, 30 seconds, 2 minutes, etc.
It is difficult to say which is better for blocking PVDF membranes for western blots as it depends on the specific application. BSA is a good choice for blocking when it is important to reduce non-specific binding, while skim milk is a good choice for blocking when it is important to increase protein detection. Therefore, it is recommended to test both options for the specific application to determine which one works better.
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