I actually think you *can *make comparisons between different proteins. We
heard a very nice talk by Jose Marquez about exactly this at the RAMC
meeting recently.
Basically, 45C seemed to be the dividing line. If your protein melts below
this it's a bad sign for crystallization and may point to
For what it's worth, we've been using thermofluor to compare the 'apparent'
melting points of enzymes with their thermal stability measured as
inhibition of their respective reactions by elevated temperature. The data
so far make sense - the differences in apparent enzyme Tm (using the same
We have proteins that melt at 60˚C but they don't crystallize. According to
your 45 degree rule we should have crystals, what are we doing wrong ?
Jürgen
On Sep 28, 2011, at 10:05 AM, Artem Evdokimov wrote:
For what it's worth, we've been using thermofluor to compare the 'apparent'
melting
Dear Raji,
what exactly do you mean when you say the melting temperature is 45deg.
Did you only test one buffer, or did you test many buffers and 45deg is
the most stable one? If you have only tested one buffer you should run a
screen testing different buffer systems (pH) and e.g. NaCl
This paper on thermofluor is a good reference and if you have access to a
real time PCR machine, different buffer systems, like the PACT screen can be
evaluated within an hour to find out the buffer in which your protein is
most stable.
It gives the Tm of your protein and if you have a high
Susan and everyone,
I should apologise for any confusion that I may have caused.
Rajiv actually asked his question a year ago, and I accidentally replied to
it a year too late!
It's an interesting question though
Patrick
On Wed, Sep 28, 2011 at 5:03 PM, Linda Schuldt lschu...@mb.au.dk wrote:
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
There is a nice paper
Comput Biol Chem. 2009 Dec;33(6):445-50. Epub 2009 Oct 20.
Predicting melting temperature directly from protein sequences.
Ku T, Lu P, Chan C, Wang T, Lai S, Lyu P, Hsiao N.
They have a list of 35 different proteins with their Tms with the references
from where they
Not sure whether this is the kind of information you are looking for:
The protein with PDB-ID 1ofc had a melting temperature of 37deg (from CD), which
was supported by the fact that it did not express in E.coli at that temperature.
At 20deg it expressed to about 60mg / (liter LB), could be
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
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
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