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I can add that in my limited experience the CD (apparent) Tm wasnt too far from that from Thermofluor, but then again they could be... (would be interesting to know if someone has done a more extensive study on the matter), but indeed why not do this (Tms) by CD or Tpr fluorescence or DSC, which would be the more standard mehtods for estimating thermodynamics of unfolding (or thermostabilty)?? That is if you want to know somehting about the protein stability overall and not just screen for conditions that are relavitely most stabilizing. Tommi On Sep 23, 2010, at 10:32 PM, Thomas J Magliery PhD wrote: > I agree that you can't take two unrelated proteins and expect their > Thermofluor Tms will be correlated with CD/DSC values. We've done > quite a bit with point mutants, and it works well for that (see an > example in our paper below). Also note that the dye is a perturbant > the reduces the apparent Tm at higher concentrations, and of course > that the whole point of using Thermofluor to help find Xtal > conditions is that the apparent Tm is sensitive to buffer, ligands, > etc. > > Tom > > http://www.chemistry.ohio-state.edu/~magliery/pdfs/LavinderMagliery2009JACS.pdf > > > On 9/23/2010 1:03 PM, Anastassis Perrakis wrote: >> Hello - >> >> The excellent paper of McCrary, uses differential scanning >> calorimetry, which will give an absolute measure of thermostability. >> >> Using Thermofluor I would be afraid you can only assess the >> relative thermostability of one protein in different conditions. >> As your fluorescence reporter would interact differently with >> exposed hydro[hobic patches in different proteins, I would be a bit >> more careful >> in comparing the Thermofluor results between different proteins ... >> I am not aware of anyone correlating differential scanning >> calorimetrywith >> Thermofluor data, but I must admit I have not looked up that >> literature recently. >> >> A. > > -- > Thomas J. Magliery, Ph.D. > Assistant Professor > Department of Chemistry > & Department of Biochemistry > The Ohio State University > 1043 Evans Laboratory > 100 West 18th Ave. > Columbus, OH 43210-1185 > > (614) 859-5743 phone (Google Voice) > (614) 292-1685 fax > [log in to unmask] > http://www.chemistry.ohio-state.edu/~magliery > Tommi Kajander, Ph.D., Docent Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 [log in to unmask]