Hey Anita,
I would like to add to Artem's comment that you can also try and cleave the
protein at 30c for 2hr and then continue the cleavage overnight at 4c (you
should check and see that your protein can withstand 30c incubation for 2hr,
of course).
In regard to your non-diffracting crystals - you can try seeding: Streak or
Macro-seed your crystals onto a screen ("screen seed") or onto the same
conditions in which the crystals grew. Sometime you might get different
morphologies that might diffract.
Good luck,
Chen--- Chen Guttman The Zarivach laboratory for Macromolecular Crystallography Building 39, Room 009B Ben-Gurion University of the Negev POBox 653 Zip Code 84105 Beer-Sheva Israel http://lifeserv.bgu.ac.il/wb/zarivach Tel. +972-8-6479519 Fax. +972-8-6472970 On Fri, Apr 8, 2011 at 04:37, anita p <crystals...@gmail.com> wrote: > Hi Crystallographers, > I am working of 23 Kda protein with a Nterminal His tag and a TEV > cleavage site. > I am getting crystals with the his tag and tev site intact, but they dont > diffract. > *Is it probable that they dont diffract because of the extra his tag and > the tev site?* > > I am trying to get rid of this tag but the reaction is optimum at 10:1 > protein to TEV ratio in micrograms overnight incubation without shaking. > I tried to run it on histrap column after this reaction but I am not able > to purify cleaved protein from TEV and uncleaved. > I have tried several times but I get 3 bands ie., the TEV, uncleaved and > Cleaved. > I have also tried to use the Nibeads instead of the histrap column, but no > difference is seen. > * Is there a possible way to approach this problem?* > > Suggestions awaited > Anita >
