Hi Monica, Your Tm results don't suggest any binding of your ligand to the protein. Your ligand concentrations are quite high so I am not convinced of even weak binding. The destabilization that you are seeing at the highest concentration may be due to ligand precipitation causing protein denaturation/unfolding.
If available, I would search for other ligands for co-crystallization. Alternatively, confirm the ligand binding by some other technique before investing a lot of time and protein in crystallization screening. John Sent from my iPad > On Feb 5, 2015, at 8:43 AM, Monica Mittal <monica.mitta...@gmail.com> wrote: > > Hi all, > I am working to crystallize a protein-ligand complex. I did a preliminary > melting curve analysis for the protein in the absence and presence of 2 > ligands (dissolved in protein buffer). I kept the other controls as buffer an > a known standard to confirm instrument performance. All expts done in > triplicates. > Now the results are like : Tm of protein alone is 56 deg, Tm in the presence > of Lig1 conc. of 0.05mM, 0.1mM, 1.0mM and 10.0mM is 56.2, 56.1, 56.0 and 53.5 > respectively !! Tm in the presence of Lig 2 conc. of 0.05mM, 0.1mM, 1.0mM, > 4.0mM and 10.0mM is 56.2, 56.1, 54.9, 52.3 and 50.6 respectively !! > Although the effective delta Tm for both is different at higher > concentration, but both are kind of making protein less stable. So i was > wondering, will it be difficult to co-crystallize them !! Any suggestions in > this regard are highly appreciated !! > > Thanks > Monica
