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 <[log in to unmask]>:
> 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
>
>
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