Hi, I'm performing ERK phosprylation assay, resolving it by western bloting. My phosphorylation buffer contents: MgCl2, HEPES, NaF, DTT, ATP, Protease inhibitor and ortho-vanadate. I understand the roles of all components except ortho-vanadate and MgCl2. Do Anyone know it? thanks

Orthovanadate is an inhibitor of protein tyrosine phosphatases

The magnesium cation is required for serene/threonine kinases to function.

FYI: Tyrosine kinases may require the manganese cation

Thanks very much!
I've been performing this assay for a few months, but I'm in trouble I'm not able to get a low ERK phosphorylation level for start. I mean,I have all my points of activation up. i tihnk I have the system saturated but I don't know how to lower it.
Have you understand me? I hope somebody can help me pleaseeeee!!!!