Hello. I am working on a kinase that binds to ATP. I cocrystallized the protein with the ATP analog AMP and solved the structure. However, while performing ITC, I am not getting a complete saturation. The attached image is for a reaction wherein a 1:10 ratio of protein and the ligand (AMP) was used. Later I also increased and decreased the ligand ratios, but there was no binding. I have also tried using different protein concentrations and repeated the experiment multiple times, but the results remain the same.
Also, the N value for one site binding is 0.01.
So, what could be the most probable reason for such a result?
My guess is that the affinity for AMP is very weak. The concentrations of protein and ligand in crystallization may be high enough to see binding, but not in ITC. Have you tried using ADP, AMPPNP, AMPPCP or ATP-gammaS?
Dear Shubhashish Chakraborty
Could you share with us concentrations and volumes you used in this titration? And how does it look like the control dilution experiment? It could be easier to answer your question then.
Regards
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