I have been running a series of ITC to look at the binding affinity of Fluoxetine Hydrochloride (Prozac) (0.5mM) with Human Serum Albumin (0.02mM). Prozac is my ligand and HSA is in my sample cell. I am using a 0.1M Tris base buffer at pH 7.4. I have been adding runs at different concentrations of ethanol (4%, 8%, 12%, 16%, in both the cell and syringe) to see if alcohol will affect binding, and the magnitude of the peaks significantly increased in the negative. Then I ran a "water into water" using buffer at 12% EtOH in both the syringe and the cell and I still was seeing large peaks. I am interpreting these as the EtOH and my buffer were reacting. But I don't understand how that could be if both the sample cell and titrant were at the same concentration of ethanol. Does anyone have any idea what could be happening? I am an undergraduate Biology major and Chemistry has not always been my strongest subject.
Isothermal Titration Calorimetry (ITC) is a powerful technique for studying intermolecular interactions, including protein–ligand binding. Let’s delve into your specific case and address the interaction between Tris buffer and ethanol in your ITC experiments.
Remember, scientific exploration often involves unexpected findings. Your curiosity and willingness to learn are commendable! Feel free to seek additional advice from experts in your field. 🌟
In my experience, it is very difficult to get the same % ethanol concentration in the ligand and protein samples for ITC experiments. Problems include: pipetting ethanol is difficult to do reproducibly (a dispensing Hamilton syringe may be the better option to deliver the ethanol);
residual solution in the ITC cell and/or syringe after cleaning; ethanol evaporation during the ITC experiment.
Dear Derek Witalec
Even simple "water to water" titration gives the signal in ITC. This is because of friction of a solution in the needle and "reorganization" of the solution molecules after moving from the solution column (restricted by the needle) to the cell. In the case of ethanol, it may produce even more heat change. What do you mean by large peaks? What is the amount of heat after each injection, and what is the injection volume?
Regards
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