Hello- Sorry to be disagreeable, but I think it depends on the protein. We've had much better success in getting reproducible crystals when we've snap frozen in liquid nitrogen in small aliquots (thin walled PCR tubes work best). We can then screen and optimize using the same protein prep- and again, it is much more reproducible than doing a fresh prep each time. Of course this is only for the few hundred proteins I've worked on and it has only been for 98% of the cases examined.
Cheers, tom ________________________________ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of David Briggs Sent: Thursday, 22 April 2010 5:27 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] degradation of protein durring freez thaw 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<mailto: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
