Hi Anita, We have had success setting up drops with TEV present. We simply added TEV at a 50:1 molar ratio and then set up the drops a couple of hours later. We went from having twinned crystals at 3A to untwinned at 2A, the crystal form also changed from orthorhombic to monoclinic, all in the same drop condition. We might have just got lucky, but it is an easy experiment to try.
Cheers Peter _______________________________________________ Dr Peter Czabotar Structural Biology Division The Walter and Eliza Hall Institute of Medical Research 1G Royal Parade, Parkville, Victoria, Australia Ph: (+61 3) 9345 2689 _______________________________________________ On Apr 8, 2011, at 12:37 PM, anita p 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 ______________________________________________________________________ The information in this email is confidential and intended solely for the addressee. You must not disclose, forward, print or use it without the permission of the sender. ______________________________________________________________________
