Hi,
The ITC protein (73kda) with ligand (GTP) showed no saturation pattern in the ITC experiment even after changing both concentrations. The pattern remains the same in the repeated experiments. Is it binding/weak binding/no binding.
It seems that it achieves saturation but you need to subtract dilution of the constituent in titrating syringe. However, from your figure it seems you have made a good fit with micromolar Kd value.
You can increase the concentrations to increase the signal/noise ratio.
You can change the temperature of ITC measurement - maybe you are measuring in such temp that enthalpy and entropy contributions cancel each other.
There MAY be weak binding. For the ITC results shown above, there is not a significant difference in the heat change/injection at the beginning of the ITC experiment, compared to the end of the experiment. One possibility is there is a mismatch between the protein and GTP solutions, in terms of salt, pH, additive concentration, etc. What buffer are you using? How are you preparing your samples? I recommend doing a dialysis or buffer exchange of your protein with the buffer you are using for ITC, then using the dialysate to prepare the GTP (from solid). Double-check the pH of both solutions. Did you do a control experiment with GTP in the syringe, and buffer in the cell? If you expect 1:1 binding, then your GTP concentration should be 10 times the molar concentration of the protein. Finally, for weak binding the ITC binding isotherms can look non-sigmoidal if the protein concentration is too low, I suggest for micromolar binding you have at least 10 uM protein in the cell
Dear Verna Frasca, if there was a mismatch between both solutions (in terms of salt, pH, etc.) I would rather expect relatively high heats per injection but it is not the case. However, I agree with you that it is important factor in well-done ITC experiment.
Tomasz Fraczyk Thank you for your answer. Actually I have used 200uM in the the syringe and 2uM in the cell. I had also tried with increased concentrations but that also showed same pattern but a sigmoidal curve. In the attached figure, the concentrations are 5uM in the cell 500uM in the syringe.
Verna Frasca i used tris nacl buffer and i ensure tahat there was buffer mismatch. Both constituents are of same pH.
I will repeat this with increasing protein concentration. what else I should do?
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