Before a buffer exchange I would increase ligand concentration, if possible. Just get the binding constant and the concentration of the protein in the crystal and CALCULATE the ligand concentration which is needed to achieve 99.9 % saturation. With moderate binding this will quickly bring you to the 100 th of mmol/l range. If this is true 10 mmol/l of ligand will never show up in the electron density.
Also: You can also try ammonium phosphate to get rid of sulfate. However, I think the problem persists because you might have now a phosphate in the active center. > Hi all, > > I have crystals of the apo enzyme growing from 1.7M ammonium sulfate > condition. After soaking with 10mM ligand (substrate analog) which has a > phosphate group, the ligand did not enter the active site of the enzyme > because of the competition of sulfate ion and phosphate group. So I am > wondering if anyone knows how to get rid of ammonium sulfate from > crystal before soaking it with the ligand solution? Thanks a lot. > > Simon PD Dr. habil. Marius Schmidt Physikdepartment E17 Technische Universitaet Muenchen James Franck Strasse 85747 Garching/Germany email: [EMAIL PROTECTED] http://users.physik.tu-muenchen.de/marius/ phone: +49-(0)89-2891-2550 fax: +49-(0)89-2891-2548
