I did remember the protein modification technique, which is reductive methylation.
-- Kendall W. Nettles, PhD Assistant Professor Department of Biochemistry The Scripps Research Institute 5353 Parkside Dr. Jupiter Fl 33458 office 561-799-8851 fax 561-799-8805 cell 561-306-7566 From: Schneider Sabine <[EMAIL PROTECTED]> Reply-To: Schneider Sabine <[EMAIL PROTECTED]> Date: Thu, 22 Feb 2007 14:08:44 -0000 To: <[email protected]> Conversation: Crystallisation of a extremly soluble protein Subject: [ccp4bb] Crystallisation of a extremly soluble protein Hi everyone, I am trying to crystallise an extremely soluble and charged protein. It is ~30kDa and has an estimated PI of 5.2 and theoretical charge over pH range 4-10 from + 24 to -29. It is still happy at a concentration of 190mg/ml and fully reconstituted with its ligand. I have tried high throughput crystallisation with 10 different screens from Nextal with concentrations of 60, 100 and 150mg/ml with no NaCl and NaCl concentrations of 100mM, 300mM and 1M in either Hepes pH 8 or Tris-HCl pH 7.5. The distribution of heavy precipitation, light crystalline precipitation and clear drops through out the screens locks like I am in the right concentration range around the 100mg/ml, but I am not getting any real hit. There are some drops with extreme phase separation. I also tried changing the temperature from 20C to 4C. I chased up a few conditions with this strong phase separation (or where I imagined little objects...) by manual screening and also adding additives like 3% Succrose, 50-200mM LiCl, 100mM EDTA, varying the PEGs (1500, 3350, 4000, 6000, 8000) as well as adding NaCl to the reservoir solution in sitting as well as hanging drop screens. But I am just getting nowhere - either just precipitation or the drop stays clear with the strong phase separation. I also re-cloned it with chopping of a few more residues on the N-term where according to a secondary structure prediction a helix starts and it is still very happy at high concentrations, but again nothing in the high-throughput screens. Has anyone any suggestions what else I could try? Thanks! Sabine This message has been checked for viruses but the contents of an attachment may still contain software viruses, which could damage your computer system: you are advised to perform your own checks. Email communications with the University of Nottingham may be monitored as permitted by UK legislation.
