Mix of Milk, BSA, Casein for membrane blocking - (May/28/2013 )

Hello,

What do you think of washing membranes with a mix of Milk, BSA, Casein instead of only one of them ? This would block more sites so why not doing it ?

Thank's

since the sites are binding non-specifically, mixing the three may just increase the concentration of blocking agent (depending on how you prepare the mixture), not fill specific sites. there will probably not be any benefit greater than just increasing the concentration of blocking agent.

on the other hand, casein and milk may cause problems if you are probing for phosphorylated proteins. milk may be a problem if you are probing for biotinylated targets.

By phosphoprotein you mean posphorylated protein ? Because it's exactly what I'm doing, why shouldn't I use milk if my antibody is specific to a phosphorylated protein ?

milk contains casein. casein is a highly phosphorylated protein.

although there are exceptions, antibodies to phosphorylated sites will bind to casein, resulting in a very high background.

unless you know, with certainty, that your antibody doesn't bind to casein (or any other phosphorylated proteins which may be in milk), it is safer to not use milk or casein to block.