My protein A is being phosphorylated and I wish to know whether it is being phosphorylated by kinase B (a well-studied Serine/Threonine kinase). Is there any other methods other than doing immunoprecipitation or kinase assay?
I would suggest kinase assay just to know whether protein A is a target for phosphorylation by Kinase B. And if it is getting hit by Kinase B, I would send it for MS analysis to know exactly h´which residues are getting targeted.
I read your question as "how can I know if my protein A is phosphorylated by kinase B or by another kinase?" If this was not the question, ignore the following.
Is there any selective inhibitor(s) of your kinase B described? If such inhibitors are available, try adding them to your experiments and see if their presence impacts phosphorylation of your protein A.
Hi! I think that the answer to your question depends directly on how much you know about the relationship between your kinase and its putative target.
The idea proposed by Sandro is the best alternative to kinase assay or IP. Nevertheless it could give you only an indication about your target phosphorylation. You can't esclude that the inhibitor blocks your target phosphorylation indirectly.
Can I ask you if you have any problem in working with purified protein or only with radiolabeled phosphate?
Many chemical kinase inhibitors can be "leaky"; depending on the concentration used they might have off-target effects and do funny things to other kinases (personal experience).
A cleaner way would be inhibiting kinase B with siRNA or CRISPR.
All the best!
So far what I have observed is when activity of kinase B goes down, my protein A degrades and I didn't observe any band shift with protein A.
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