Re: [ccp4bb] Protein preps become a jelly

[Roger Rowlett]

Not necessarily uncommon. To minimize protein-protein interactions that might be causing gelation, you might change the pH of the solution so that is is not close to the pI, use a small amount of chelator to sequester metal ions, and maintain a modest ionic strength. Other additives that might stabilize soluble protein would include glycerol or other polyols, but these may interfere with crystallization. A typical storage buffer for us is 20 mM Tris-Cl, pH 8.0, 10 uM EDTA, 100 mM NaCl. You can add 5-50% glycerol to stabilize protein if desired, and/or omit the NaCl if you protein is tolerant. It is also possible your protein is crosslinking via disulfide bond formation, in which case a modest amount of reducing agent may be necessary, e.g. 10-100 mM beta-ME, 1-10 mM DTT, or a small amount of TCEP. Usually, the less stuff you put in the storage buffer, the easier it is to crystallize the protein, but as always, YMMV. Cheers, _______________________________________ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 8/30/2011 11:31 AM, aidong 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/