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.
On 23 Sep 2010, at 18:40, Philippe DUMAS wrote:
Le 23/09/2010 17:28, Raji Edayathumangalam a écrit :
Raji
I suggest having a look to this paper:
McCrary et al. J. Mol. Biol. 264(1996) 784
where you will find an interesting study on protein stability and an
interesting comparison with other proteins.
Philippe Dumas
Hi Folks,
Sorry for the pre-xtallo question; pre-xtallo right now, but hoping to
take my protein the xtallo way one of these days!
I am currently performing Thermofluor assays with my protein and the
results show that the Tm is ~45C. I am looking for some examples of
proteins and their melting temperatures so that I can gauge where my
protein falls in the spectrum of unstable-to-stably folded. For
example, the melting temperature of some forms of lysozyme is 73.8C
(very stable, I suppose).
Just need a sense for whether my protein is considered unstable or
somewhat stable. Please could you share some examples.
Many thanks.
Raji
-----------
Raji Edayathumangalam
Joint Research Fellow
Harvard Medical School/
Brigham and Women's Hospital
Brandeis University
<McCrary-JMB264(1996)784.pdf><p_dumas.vcf>
--
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
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