Dear Jobi,
The usual suspects are:
- add another purification step. In fact I'd do that first:
* Ni-column
* remove His-tag
* Ni-column
* gel filtration
if you have done that: add another step to test for better crystals
- additive screens
- room temperature data set to check if freezing hampers the crystal
- seeding
- crystallisation at different temperatures
- removal of the His-tag (or leave it on if it is cleaved)
etc, etc, pp
Cheers, Tim
On Wed, Apr 13, 2011 at 05:44:12PM +0800, Jobichen Chacko wrote:
> Dear All,
> We got crystals for a 35 KDa protein with 323aa including His tag and
> linker. It was originally crystallized in 0.1M BisTris Propane pH:6.5, 0.2M
> Potassium thiocyanate and 20% PEG 3350. Later we managed to obtain crystals
> with 0.1M BisTris pH:6.5, 0.2M Potassium thiocyanate and 20% PEG 3350 as
> well. Crystals are 3 dimensional in shape and 0.2-0.3mm long. Maximum
> resolution obtained till now is 5.8Å. Tried various cryo conditions like
> Oil, glycerol, salt and sugars. However, the resolution hasn't improved. The
> crystal tends to break in the presence of glycerol.
> Kindly give your suggestions to improve the resolution.
> Thanks.
> Jobi
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Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen
phone: +49 (0)551 39 22149
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