Dear Anita, Sometimes the protein of interest has a relatively strong inherent binding affinity to the IMAC column. Have you tried to bind the cleavage reaction to an IMAC column and then elute using a shallow imidazole gradient?
In fact, Porath developed IMAC chromatography as a tool for protein fractionation long before molecular biology provided the tools necessary to add poly-histidine tags to target proteins (Nature. 1975 Dec 18;258(5536):598-9). Best regards, Martin On Apr 8, 2011, at 4:37 AM, 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
