I would like to know which is prefer BSA or nonfat dry milk to block nitrocellulose membrane.
BSA is a more expensive, but more powerful blocking buffer. I use 3% BSA for about 1.5 hours. You can also use 5% non-fat dry milk as an in-expensive alternative, but I would recommend increasing the blocking time. If you're using milk, make sure to filter through a 70um filter before use to prevent streaks.
Hi there I basically agree with the above. Having performed many Western blots I would either block with 3-5% powdered milk at room temp for 1 hour or at 4C overnight. In my experience both lead to successful blocking. I have heard it said that supplementing 3% powdered milk with 1% BSA can reduce background further but for blocking times of more than 1 hour can also reduce specific binding
Hello
i agree with researcher , also you can block nitrocellulose membranes with 5% milk in TBST for 1 hour at RT and wash 3 x 5 m , OR block with TBS buffer/1% BSA for 1 hour at 37°C or overnight at 4°C.
add , There are a similar discussion about this issue in RG :
Good Luck
https://www.researchgate.net/post/What_is_the_difference_between_BSA_and_Non-Fat_milk_NFM_blocking_of_the_membranes
https://www.researchgate.net/post/What_is_the_difference_between_non-fat_milk_and_BSA_for_primary_antibody_in_western_blot
Hello there! Personally, I think both of the choice are fine. I used both 5% non-fat milk and 3 % BSA in TBST for blocking for 1 hr before, and there are no significant differentce I think. But BSA is much expensive, so I guess it depends on your situation. Good Luck!
Ultimately, it depends on your antibody. For 90% of my applications, I block for 10 minutes in 5% milk in PBST. But there are certainly antibodies for which milk is a terrible idea, and you need to BSA or perhaps serum. Best of luck.
A follow up question: from an economic perspective is it best to block with 5% NFDM (although for a longer period of time), unless you are very certain that your antibodies don't prefer milk? Is there also a rule of thumb which kind of antibodies are sensitive for NFDM blocking?
Milk will work for most things. However phospho epitopes can be masked, especially phospho-tyrosine. You'll also need to use TBS as opposed to PBS based buffers for phospho-epitopes. Again, I routinely block for 5 - 10 minutes.
As a last cost saving tip, which you are probably doing, make up your antibodies in PBST (or TBST) with a little Sodium Azide. I usually make 10 mL at a time. You can reuse this for months and months if kept at 4 degrees. Just wash the milk off of your blot after blocking, or you will get milk precipitates in your Ab solution.
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