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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 -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A