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 > > > > >
