Dear Sonia, Have you passed the protein through Size exclusion column and confirmed that it contains a single oligomer? Do you use fresh protein preparation for ITC? If you have tried the above two, then increase the protein concentration. Checking at diff. pH buffer or temperature can also refine your data to a great extent.
good luck On Mon, Aug 4, 2014 at 12:14 PM, Sonia Majumdar <[email protected]> wrote: > 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. > -- rashmi
