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.
Why do you say the saturation point is not reached?
The strength of the interaction is indicated by the association/dissociation constant.
Very likely you are misled by the fact that the background injection heat effect (usually called "dilution heat") is considerable (almost 20 kcal/mol).
You must think on the parameters obtained from the fitting, whether or not they are reasonable. For example, an interaction enthalpy of 80 kcal/mol is quite large, but for a DNA-protein interaction it might be reasonable and also the buffer may contribute to amplifying the overall enthalpy. Another issue is the fractional stoichiometry (or fraction of active protein), with a value of 1.7. Do you expect the protein to bind several DNA molecules? Have you measured the protein concentration by UV-absorbance or by colorimetric methods? Have you determined the DNA concentration by UV-absorbance?
Have you done the appropriate controls? E.g., DNA into buffer, buffer into protein, experiments at different temperatures...
Why do you say the saturation point is not reached?
The strength of the interaction is indicated by the association/dissociation constant.
Very likely you are misled by the fact that the background injection heat effect (usually called "dilution heat") is considerable (almost 20 kcal/mol).
You must think on the parameters obtained from the fitting, whether or not they are reasonable. For example, an interaction enthalpy of 80 kcal/mol is quite large, but for a DNA-protein interaction it might be reasonable and also the buffer may contribute to amplifying the overall enthalpy. Another issue is the fractional stoichiometry (or fraction of active protein), with a value of 1.7. Do you expect the protein to bind several DNA molecules? Have you measured the protein concentration by UV-absorbance or by colorimetric methods? Have you determined the DNA concentration by UV-absorbance?
Have you done the appropriate controls? E.g., DNA into buffer, buffer into protein, experiments at different temperatures...
I think, as Adrian, that the thermogram is dominated by an enthalpy of dilution or another interaction which seems to be constant over the injections. The error bars are so large that the model fitting is not valid.
Yes, errors are big, but, if control experiments are fine, I would say there is interaction. A global fitting with several titrations will help in reducing the errors
Adrian Velazquez-Campoy and Jean-François Gal thanks for your valuable answers.
I have done the ITC again at 20 ℃. This time the thermogram is better than the above, the last peak is 2.5 times less than the second peak (largest peak). The values I got are N (sites)=0.89±0.29. The Kd value is 8.3±4.34 µM. The interaction enthalpy is similar to the above. The concentration of the protein is 7 µM and that of DNA is 140 µM. I have measured the concentrations through nanodrop.
Further I did controls, buffer into protein and DNA into buffer. They show uniform peak patterns with 0.1 degrees change in DP. In fact, I checked the negative control of my experiment, the mutant protein, The DP change is less than 0.2.
Now my questions are,
1. Do you think the errors are still large for my positive experiment?. That is the best I could produce after repeating and adjusting concentrations several times.
2.The thermogram of my negative controls seem fine but the data can not fit into interaction isotherm. The Kd is smaller and with large errors and N sites value is more than 2 with large errors. Do you think it can be repaired.
3. As far as my understanding about the Kd is that Kd value gives rise to c value, c=n(M)/Kd. So the less the Kd from the concentration of the protein, the stronger the interaction. The more the Kd from the concentration of the protein, the weak the interaction. Please let me know if I am correct. Thanks.
The c value is slightly lower than 1 (7/8.3) and, therefore, you cannot observe a nice inflection point in the isotherm.
The errors can be reduced in several ways. You can do several titrations a fit them globally, or fit them individually and calculate averages and standard deviations.
The error you get for the Kd is large, but reasonable.
The negative controls, if everything is fine (or the ligand has low dilution enthalpy or/and does not self-associate), should not reflect anything close to an interaction. Thus, they should exhibit small and similar heat effects. Those negatives controls are not to be fitted with an interaction model.
As you say, the c parameter and the Kd are related. The higher c, the lower Kd, the higher the sigmoidicity, and the higher the affinity. But the protein concentration makes a diiference in c; that way, two experiments with different affinities may have the same c value.
I agree as Adrian explained and you can go for a number of experiments in the different ration and concentration that will help you to access your thermogram as well as data obtained from that .
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