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High background using 5% BSA for blocking Western Blot - (Dec/09/2010 )

I compared blocking my nitrocellulose membrane with 1% BSA and 5% BSA. I found that a higher percentage of BSA caused a higher background signal on my membrane. Do you have any idea why that it?Will 5% skimmed milk will be better as a blocking agent?
Thanks a lot!


In general, I prefer 4% milk in TBST over 5% BSA unless there is some reason I have to use BSA (such as for phospho-epitope antibodies). If I do need to use BSA, I block overnight instead of for the 1-2 hours that I block with milk.


In my experience as well, milk is a better blocking agent than BSA. We also observe that some antibodies generally give a high background. Did you observe this in different experiments or just that one?


Milk is also no good for alkaline phospatase conjugated antibodies as the milk contains phosphatases leading to increasing your background. Milk is fine for HRP though.