Hello.
I'm working on check protein-protein interaction.
My hypothesis is protein A (kinase) phosphorylates protein B.
So I tried lot of Co-IP bur failed. So I planned to pull-down assay using tagged-protein A and tag antibody conjugated agarose bead.
In this case, I wondered the addition of ATP is required for Co-IP or pull-down assay or not. Is kinase usually form protein-protein interation structure and don't get off from each other?
And someone can recommend protocol of pull-down assay or reference paper?
I appreciate to read mine and hope you have a nice day!
Kinase/target interactions are not always high-affinity: a kinase that sticks to its target with a low dissociation constant is not a kinase that can go off and phosphorylate other targets, after all. No amplification cascade.
Consequently, you may have an interaction, but the interaction is never maintained long enough for a Co-IP to be successful. Adding ATP probably won't help because now your transient (unproductive) kinase reaction will become a transient (productive) kinase reaction, and the phosphorylated site might be even lower affinity for your kinase.
You could include low concentrations of crosslinkers, possibly.
Or try a different approach, such as kinase assays? Since you already apparently have recombinant protein A, you're well on your way toward doing exclusively in vitro stuff anyway.
Do you have phospho/non-phospho antibodies for your target protein B? That would be another option.
Possibly the kinase undergoes a conformational change when ATP is available and only then can bind to its target. ATP then gets hydrolyzed quickly while the phosphate is transferred, and you won't find anything in your co-IP.
What therefore *could* work is using a sulfur analog of ATP, ATP-gamma-S instead. It probably will bind, but not be hydrolyzed and thus stabilize the protein complex.
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