I have used protein conc. from 20uM upto 60uM. the protein to ligand ratio used is 1:10 and also 1:5 has been tried. I have tried Tris-Cl 50mM pH 7.0 with 150mM NaCl and 2mM MgCl2, Also HEPES 20mM pH 7.0 with 150mM NaCl and 2mM MgCl2 has been tried. There is no increase in the heat change even when protein is increased and at still higher conc. it precipitates. The temperatures tried are 30C, 25C, 20C. below 25 there is no binding (heat change is more but in subsequent injections no saturation is achieved). Thanks and regards
On Mon, Aug 4, 2014 at 4:38 PM, Rashmi Panigrahi < [email protected]> wrote: > 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 >
