However, if your protein is not a protein tyrosine kinase, you may check your western condition.
Yes, this is an essential control. Use lots of Lambda protein phosphatase to "dephosporylate" your protein and us the resulting material as a negative control in Western.
My experience is that phosphorylations of over-expressed proteins in E. coli are always auto-phosphorylation.
Should be the case when there is tons of overexpression. But to be fair, bacteria (and E.coli) do possess tyrosine kinases. It's still possible that one of them does work on some sites found in your protein. Dima
