Dear all
I have performed ITC for protein and ligand solution.generally we substract heat of dilution of ligand from overall heat for ligand titration into HSA .is it necessary to substract the heat of dilution of protein solution from overall heat of titration?
Hi! I have experience with nano-ITC experiments involving inorganic compounds, not proteins, but there should be no differences.. For each titration, I only subtract the heat of the titrant dilution (the lingand in your case), but never that for the titrand dilution. When you inject for instance 10uL in 1mL solution (for nICT), the heat involved in the dilution of the 10uL in 1mL is not negligible, whereas the protein solution changes its volume from 1000uL to 1010uL. You don't have to consider the protein dilution
It depends on your system and on the binding enthalpy. If you have enough protein, perform a blanc-in-protein titration to be sure that this heat is negligible. This is mostly but not always the case.
Sorry but I disagree.. you should not have any binding.. what do you mean with "blanc-in-protein"? I guess water (or buffer) into the protein solution (please correct me if I am wrong).. you don't have any binding.. it is only an effect of dilution
Yes, exactly. You should just see the effect of dilution, which should be small compared to the actual titrations heats and therefore it should be negligible. This is in most cases true, but not in each and every case. Therefore, in order to be sure that the dilution of the protein is a negligible effect, it is a good practice to perform always also the titration of your buffer (or water) into the protein (except if there are very good reasons not to perform it).
It also a good practice to perform the buffer-into-buffer titration. Ideally, no heat should be detected. This is however never the case, and it allows to detect instrument malfunctions.
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