I´m working on a protein that becomes phosphorylated following DNA damage. From in vivo results using phosphorylation mutants we can say the phosphorylation is sequential at two sites.
We identified the kinases acting on both sites using kinase inhibitors and Ipp.
My question is, how is it possible that when I perfomed in vitro kinase assays using the kinase acting at the second phosphorylation residue I get a phosphorylated band although the first residue is not modificated (the phosphorylation is sequential as concluded by in vivo results).
Thank for yout time.