I am using MicroCal ITC 200 (GE) and performing an interaction study between a DNA aptamer (12 kDa) and a peptide ligand (1.91 kDa). How can I explain an ITC curve with both negative and positive peaks (exothermic and enothermic peaks) at the same time? What type of different mechanisms can be involved in the binding process?
Looking forward help. Thanks a lot.
The initial exothermic reactions may be due to the salt concentration gradient that is used for disolving your samples. you may raise the concentration of peptide or DNA to get good results. If you are using the same solvent to disolve them. Have you subtracted the dilution factor? What is the overall heat change ?
Thank You for your reply.
Yes I tried with higher concentration of the peptide ligand and got an abrupt saturation in the heat burst curve (file attached below). So I tried using a lower concentration of the ligand, keeping the aptamer concentration constant in both the expts.
It seems reaction is completed within 10mins. Most of the biological samples are not stable at 37 degrees. Most of the people store the bio samples at ice temperature. when you suddenly change the temperature in the ITC machine to higher degrees the sample may undergo denaturation. I would recomend to conduct the experiment at low temperature - May be at 25 degrees. Also reduce the aliquotes of sample addition to 0.5 micro liter per injection. you can capture the heat change of proteins samples at low temperature.
I would suggest to reduce the additions to 0.2microliters by increasing the number of injections. also reduce the time gap between injections. so that you will see beautiful thermogram
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