How to process the protein and ligand Isothermal Titration Calorimetry (ITC)?

The two materials are very complicated polysaccharide and proteins. So, vibration is somehow expected. However, after each exothermic reaction, an endothermic one is occurring; which I do not have any idea about. Moreover, the baseline is shifting down. I am wondering if I am allowed to normalized the baseline after subtracting the control data? if yes, how?

I am using isothermal titration calorimetry to measure the binding affinity and stoichiometry between a small molecular weight poly(amino acid) (3500 - 4500 Da) (SYRINGE) and high molecular weight glycosaminoglycan (80,000 - 100,000 Da) (CELL). I have used the experimental design function on NanoAnalyze to optimise the syringe and cell concentration to yield a sigmoidal curve with an assumed stoichiometry of 10 (n=10). However, after running the experiment, I obtained the thermogram attached below. I have also attached the binding isotherm, which shows high standard deviation of the KD, delta H and n values, even after excluding the outliers. I have tried multiple experiments to optimise the concentrations and injection volumes. What can I do to improve this result?

The heat of dilution of the buffer has been excluded in this experiment. The amplitudes of the outer and inner values of each peak follow opposite trends! See the attached figure.

（Protein-Polysaccharide interaction in PBS buffer）

I tired different concentrations of Ligand (DNA) and protein, (400 µM, 30 µM), (200 µM, 10 µM),(200 µM, 5 µM), (100 µM, 5 µM). The data is improved but still the saturation point does not reach (File attached here). Does this indicate that the interaction is weak or there is no interaction?. Your suggestions will be highly appreciated.

 the amplitude of exo- and endo- values are following the same trend for each peak! see attached figure.

(Protein-protein interaction in Tris buffer containing 10% glycerol)

Hello,

I am doing ITC and getting repeatable curves that look very promising. After reading some articles, it seems the biggest challenge to avoid is buffer mismatch. Is there a way to rule out that my curve is indeed ligand to macromolecule binding rather than buffer mismatch (i.e. shape/values of curve, shape/values of trace, thermodynamic values)?

I am very careful when preparing my samples but because my ligand is a small molecule I can't dialyze. I am pretty certain my buffers are well matched but I just want to be certain.

Any help would be appreciated.

Hello,

I am doing a titration of ligand into protein and am getting good heat releases compared to my ligand into buffer.

I am getting more heat release in later injections. Could cooperativity explain this because I have used varying concentrations of protein and ligand and get the same curve.

Thank you.

Hello, I'm Sanam , a grad student new to ITC. I am trying to do titration between a protein and small molecules that we knew it binds to the protein since it's a commercial inhibitor. I purified target protein . For doing titration the protein concentration was 0.0501 mM( determined by uv absorbance at 280 nm and extension coefficient), and compound concentration was 0.59 mM. However, I got that result (attached file). I will really appreciate if someone could guide me a d help me. I am so new in ITC. By the way, I used TA affinity ITC. Thanks in advance.

Hi,

The ITC protein with ligand 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.