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
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> I apolozize an off topic query.
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> 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.
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> Thanks in advance
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> Thomas
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