Re: [ccp4bb] ITC thermogram
Probably, you have two mode binding between your ligand and protein. First time, when you had approximately 7 times molar excess of ligand, you could not monitor the endothermic reaction. When you went down to approx 2.5x lower conc of ligand, you could monitor endothermic reaction. However, what is Kd of the reaction? Is it very strong binding? How much micro cal values you are getting? When you dont see saturation, you increase the conc of ligand, why did you go down?
Re: [ccp4bb] ITC thermogram
Hi, I think the ligand concentration (250nM) is too low for the titration to reach saturation. To get properly saturated thermogram people sugest to use the ligand concentration 15-20 times higher than the macromolecule. On reducing the the ligand concentration you said you have obtained endothermic thermogram. Have you checked if the dP values are comparable or not? Sometimes when using too low concentrations of both protein and ligand may result in giving this kind of results, since the signals for the binding reactions get over-rided by the noise of the experiment. Running a blank experiment may help you to conclude about this confusion. Madhuparna. -- Madhuparna Bose. Senior Research Fellow. Department of Biotechnology. IIT Kharagpur. West Bengal. India. On Mon, Dec 15, 2014 at 11:35 AM, sajid akthar b_sajid_...@yahoo.co.in wrote: Dear All I am running ITC experiment for my protein. At 250 nm Ligand concentration (protein was 40nm) I was getting exothermic thermogram. But it was not saturated. I reduced Ligand concentration to 15 nm and checked again. I got endothermic thermogram. I dont understand why I am getting both endothermic and exothermic thermogram for same protein and ligand. Can some one explain Thank you Sajid
[ccp4bb] ITC thermogram
Dear All I am running ITC experiment for my protein. At 250 nm Ligand concentration (protein was 40nm) I was getting exothermic thermogram. But it was not saturated. I reduced Ligand concentration to 15 nm and checked again. I got endothermic thermogram. I dont understand why I am getting both endothermic and exothermic thermogram for same protein and ligand. Can some one explain Thank you Sajid
