Hi, Obvious answer - don't freeze it. If you cannot set your crystallisation screens up straight away (the preferred option, IMHO), why not try leaving the protein at 4C overnight? Does it still degrade?
Freezing & thawing a protein-protein complex not a good idea, I think. Hth, Dave -- Delivered via an Android. On Apr 22, 2010 6:55 AM, "Jhon Thomas" <jhon1.tho...@gmail.com> 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
