Lots of things change with temperature. An excellent book on the
subject is P. Dohzu's "Cryobiochemistry" (1977) Academic Press, which I
think every lab that freezes biological molecules should have on hand
($15 on Amazon.com). For example, did you know that the pH of a tris
buffer jumps from ~8 to ~14 when you cool it from 300K to 77K?
Probably one of the few cases of a well-characterized freeze-thaw effect
on a protein-protein interaction is the diphtheria toxin dimer: Steere,
B. & Eisenberg, D. (2000)."Characterization of High-Order Diphtheria
Toxin Oligomers", Biochemistry 39, 15901-15909. At least, that's the
one that leaps to mind.
I agree that a single data point does not a trend make, but a single
well-characterized case (as opposed to an annecdote) can lead to insight.
-James Holton
MAD Scientist
Mischa Machius wrote:
A couple more thoughts:
1. thermodynamics says that proteins denature at low temperatures just as they
do at high temperatures.
2. flash-cooling does away with some of what thermodynamics says (not an
equilibrium process anymore)
3. Whether a given protein can be frozen needs to be experimentally
demonstrated before accepting such a step. In many cases, the protein won't
denature, but it may well aggregate.
4. In the particular case discussed here there is another aspect. Tris has a
Delta(pH)/Delta(T) of -0.03. This means that freezing a protein at -80°C may
well move the pH up by 3 units, which may or may not be tolerable. So it is
important what Tom said earlier in the thread: spend some time working out the
buffer.
Cheers! MM
On Apr 22, 2010, at 1:54 AM, Jhon Thomas wrote:
Hello BB
I apolozize an off topic query.
I am working with small proetin-protein complex of 24kDa. I purify this
N-terminal His-tagged complex through tylon resin in 20mM of Tris pH-8.0, 0.3M
NaCl . After purification this protein complex are dialysed in 20mM tris
pH=8.0.I am able to purify enough amount of protein for crystallisation, which
can be concentrated upto 10mg per ml. Then i check the dgradation on the
polyacrylamide gel after concentration of the protein. I donot see any
degdation protein band on the gel. I store the protein at -80 in aliquotes of
100ul immedaitely after concentration in same buffer. protein concentartion are
done at 4 degree by centrifugation. Next day before setting up the trays for
crystallisation screening, protein solution concentration check is being done.
it turns out that this complex has degraded and concentration is only 1-2 mg
per ml. i would appreciate the suggestions to prevent the degradation of
complex or How should i make it more stable? so, that i can proceed for the
crystallisation. I would really appreciate the suggestions.
Thanks in advance
Thomas
