Dear Sonia Your value of 0,1ucal/sec is weak but if you can determine the intial and final plateau and the slope at the transition, it may be ok. Triplicate experiments will give you an idea of the quality of your measurements. Similarly, the control experiments with only the ligand injected in the dialysis buffer and/or with a mutant of your GTPase will help you to evaluate the quality of your preliminary data Otherwise, you can vary the following parameters
1- Protein concentration in cell: Increasing the protein concentration should increase the signal and improve the determination of the slope (classical parameter changed in ITC and called the c factor, see Ropars, V et al 2011 PNAS for an example) 2- Temperature: you can have higher heat exchange at a lower temperature (see Czarny, B 2013 J Med Chem) 3- pH and buffer: Similarly,measurements varying these parameters may change the thermograms and will give energetics information (see Czarny, B 2013 J Med Chem) For the n value of 0.33 Is your enzyme a monomer? Such n value may be related to stoichiometries of 1 ligand to a trimer of protein (or a dimer if n is not well defined) It can also be due to a weak fraction of protein active due to the purification protocol. It is interesting to follow the n value with different preparation of proteins. N can vary with proteins just issued from last purification step or with older proteins Hope it can help Regards JB De : CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] De la part de Sonia Majumdar Envoyé : lundi 4 août 2014 08:45 À : CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] offtopic: related to protein purification Dear all, I am working on a GTPase with two tandem G-domains. While doing ITC experiments the heat change obtained is very poor 0.1ucal/sec, however the ITC profile is showing gradual saturation. The n value is very poor 0.33. I presumed the protein has bound GDP and tried to separate the nucleotide bound and unbound forms using Mono-Q. 3 peaks were obtained containing the same protein. However, ITC done with the proteins corresponding to the 3 peaks gave the same results. Please suggest what could the three peaks possibly mean and how to modify the ITC experiments. Thanks in advance. Regards.
