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
