Sample cell was 0.097mM and syringe (1.25, 2.5, 5, 31.25 125mM). At125mM,it was endothermic process and became exothermic at lower concentrations . and enthalphy decreases with the decrease in concentration wheras entrophy was positive in all cases.
If you are studying a 1:1 interaction and the concentration in the cell is around 0.1 mM, the concetration in the syringe should not be higher than 1-2 mM.
Mathematically, the binding isotherm will be sigmoidal (that is, there will be an inflectin point) only if c > 1, where c = Ka x [P]cell = [P]cell / Kd. Absence of inflection point does not mean there is no interaction, but that the interaction affinity or the concentration of protein in the cell are low.
The observation of an enthalpy decreasing with the concentration of ligand in the syringe suggests you are mainly observing ligand dilution when you employ high ligand concentrations, a process that very likely obliterates the protein-liagand interaction (if it exists).
As in many cases of people asking for advice on experimental results, showing an image of the experimental results will surely help in figuring out what is happening.
Thanks Adrian for a clear and concise answer. As Adrian suggests, there are some experimental details one can add to help complete the picture. This is what I recommend: try looking if your binding entity in the syringe is actually still dissolved and not crashed out of solution by using Dynamic Light Scattering (DLS), (make sure to use the same buffer conditions as your ITC set-up) and use NMR to look at how soluble it is. You can also use DLS to QC your protein solution too, however, MALS will be more sensitive. Once you have figured out if both entities are not aggregating and are soluble, try the experiment again using the proper concentration ranges. If you can't work at these concentration ranges, try reverse ITC. There are many protocols online on how to do that. Good Luck and hang in there! Biophysics is never easy.
Thank you Adrian and Christine for your valuable suggestions. I repeated the experiment with protein 0.1mM and ligand (denaturant) 1mM. Below are the attached files of raw heat data and the data after fitting by independent model. Is the curve like this because the protein is denatured or is it because of experimental or fitting errors. Hoping for your kind suggestions. I am a beginner in ITC and really lost. I am not sure about appropriate models selection and data fitting.
The ligand you are injecting is a denaturant? Which denaturant? It is tricky to perform titrations with denaturants. They usually are effective at concentrations larger than 1 M and proteins have many (vaguely defined) interaction sites for them. Considering a cell volume of 0.2 mL, an injection volume of 2 microL, and an intended concentration of 3 M ligand after injection number 10 (that is, more or less at half titration), the concentration of ligand in the syringe must be 30 M. This is totally impractical.
An alternative approach you may follow is performing the reverse titration Christine suggested. In that case you must place the ligand (denaturant) in the cell at high concentration and then inject the protein. The problem is that you will get huge background heats due to dilution of the ligand (even a small injection will result in a large heat effect).
Guanidine hydrochloride is insoluble above 6 M (at room temperature), it has a large dilution heat and it has very small association constant with proteins. Therefore, it is not the best scenario...
Thank u @ Adrian for ur perfect and valuable answer. i think u r realy expert in ITC
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