BSA vs Milk - (Aug/26/2011 )

I have a question about blocking.

When I use 5% milk to block, I usually see no or very faint band. But when I use 5% BSA to block, I usually see band of interest plus some non-specific bands. Does anyone know why?

Perhaps there is something in the milk that is binding your antibody... this is especially likely if you are using a phospho-specific antibody, as milk contains lots of phospho proteins.

We have found that for our antibodies using Milk for the blocking step and secondary antibody dilution buffer and BSA for the primary antibody dilution buffer gives the best results. Using milk for all of the steps can sometimes block the specific signal. BSA is a weaker blocking reagent but using it for all of the steps can lead to non-specific background.
-Julie Diamond, Cell Signaling Technology Product Scientist