Le Jeudi 9 Août 2012 09:55 CEST, rashmi panigrahi <rashmi.panigrah...@gmail.com> a écrit:
Hello Rashi There is no problem with ITC at "low temperature", apart for a likely slower binding and, hence, lower heat power signal. Do not conclude that there is no binding if deltaH is close to 0 around 20-25 °C. You may well have DeltaH = 0 at a given temperature, and yet the affinity constant is maximum at that temperature (by the Van't Hoff equation). However, if you have no signal (DeltaH = 0) at several temperatures, this means that this peptide does not bind. It is very well established that DeltaCp = dDeltaH/dT is often large, which means that a 15°C variation is sufficient to get a significant change in deltaH. Conclusion: do ITC at low temperature and try to increase as much as possible the concentrations of the protein and of the peptide. Also, if the peptide is too hydrophobic, you can put it in the cell and the protein in the syringe. Finally, you may also try to repeat CD in presence of various amounts of peptide and see if this results in Tm increase. Philippe Dumas > Hi All, > I am working on a protein that has a Tm of 30 degrees, and by CD I have > observed that the secondary structure is intact at 10 degrees and slowly > starts unfolding at 20 degrees. > Literature suggest that it binds to a 5 residue peptide. I tried doing ITC > @ 25 and 20 degrees , there was no binding observed. > > Does any one have the experience of doing ITC or Biacore(SPR) at 10 or 15 > degrees? > wondering that temperature is a problem as suggested by CD. > thanks for your suggestions > > > -- > rashmi
