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
magli...@chemistry.ohio-state.edu
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
tommi.kajan...@helsinki.fi
