Before a buffer exchange I would increase ligand concentration,
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.
PD Dr. habil. Marius Schmidt
Technische Universitaet Muenchen
James Franck Strasse
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