Hello Sabine, Maybe this is an option:
Structure, Vol 14, 1617-1622, November 2006 Lysine Methylation as a Routine Rescue Strategy for Protein Crystallization best regards & good luck Christian Schneider Sabine wrote: > > 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. >
