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
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