I had a similar problem with crystals that were obtained from PEG-based precipitants over long periods of time. If I harvested the crystals in about 5 days, they would diffract to ~3 Angstrom. If I let them grow any time past about 7 days, they became PEG-alated and didn't diffract at all. I personally have not had that happen in salt-based precipitant conditions. I would try adding up to 10mM DTT or 5mM TCEP to minimize proteinaceous skin formation, which I believed was crosslinked by the PEG. Attempts to change PEG's did not work and I couldn't find a salt based precipitant condition that would give us crystals where the active site was not occluded by the salt.
BTW, have you purified the prep over SEC followed by IEX or RP columns? Have you dissolved the gel and run mass spec to see if there is any proteolysis going on? If you have an active proteolytic fragment, there may be one or more hydrophobic patches being exposed and this could cause the protein to gum up in order to bury those patches. Anyway, good luck! Bryan From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Laurie Betts Sent: Tuesday, August 30, 2011 11:41 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Protein preps become a jelly I have had protein crystals (so very high protein concentration) that turn into gummy bear-like objects, where instead of crumbling they are like, well, a gummy bear or a piece of rubber. I attributed it to oxidation or other chemical ageing processes. I am sure others will have suggestions for preventing this. On Tue, Aug 30, 2011 at 11:31 AM, aidong <a...@xmu.edu.cn> wrote: Dear Buddies, Sorry for bothering you with an off-ccp4 question. We recently are experiencing a very strange phenomena. A couple of protein preps with reasonably high concentration (10-20mg/ml) become a jelly after storages for overnight or a couple of days at 4C. All of them have been purified by gel filtration. Some of these proteins behave like this from very first preps but some of them had been very kind to us previously. We have googled extensively in CCP4BB and www but it appears this only happens to us. It would be highly appreciated that you could exchange their experiences or provide your suggestions. Aidong Han, Ph.D Department of Biomedical Sciences School of Life Sciences Xiamen University Xiamen, Fujian 361005 China Phone: 0592-218-8172 (O) 0592-218-8173 (L) Web: http://life.xmu.edu.cn/adhanlab/ -------------------------------------------------------------------------- Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
