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What exactly does BSA do in immunohistochemistry? - (Jul/24/2011 )

I am labeling HeLa cells with a peptide-quantum dot conjugate (which will bind to an integrin on the cell surface) and following the standard immunohistochemistry protocols (blocking, fixing, etc). But I can't find a good explanation of what blocking with BSA actually does. I know it contains proteins and stops non-specific binding. But how exactly does it do this? Thanks!


Basically it contains proteins that bind to other proteins, or will be bound to proteins that like to scavenge proteins from solution. This means that your antibody will not be scavenged by these proteins, and thereby lowers background.


If you just added your antibody directly to your sample, it would bind to your protein of interest, but it would also bind to other proteins. There are many proteins in the body which are quite 'sticky', meaning they will bind non-specifically to lots of things, including antibodies. This means that your antibody will label not just your integrin of interest, but also other random non-specific things, in background staining.

Blocking with BSA first means that all these non-specific sticky proteins are saturated with the high concentration of BSA that you have added, leaving your antibody free to bind specifically to your protein of interest. BSA is used mostly because it is cheap and abundent, people often also use plain old milk powder from the local cornershop as it contains a mixture of random nonspecific proteins found in milk, good for blocking in ELISAs and such. (Although I guess you wouldn't use opaque milk powder for highly sensitive immunohistochemistry)