Hi Sabine, I vaguely recall that an old colleague had used a mini detergent screen (as additive) to solve a problem such as yours. It may be worth setting up a plate or two to screen a few conditions before you embark into more molbio. You can use the 96 well plates with multiple wells (Corning sells plates with 5 depressions in sitting drop geomtery). In that way you could screen up to five detergents per condition.
best of luck Savvas PS. A broad additive screen may also be worth a shot for that matter. Quoting Schneider Sabine <[EMAIL PROTECTED]>: > 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. > > --
