Thanks everyone for your suggestions ! Artem has pointed out that low diffraction of the crystal might be because of other problems .. If you could* highlight a bit more on this issue it would be helpful for me.* I have tried to seperate the cleaved, uncleaved and TEV over MonoQ column but there was a single peak and all of them came out together, there was no seperation. I even tried to cleave the protein at 30 degress and it starts precipitating. I have also tried binding it to the IMAC as Martin has suggested but then I get a single peak while running the imidazole gradient and its tev, cleaved and uncleaved together. And I also get the flowthrough while loading unto the column which should be theoritically the cleaved one but it is a combination of cleaved uncleaved and tev.
awaiting for bit more suggestions Anita On Fri, Apr 8, 2011 at 10:48 AM, Artem Evdokimov <artem.evdoki...@gmail.com>wrote: > For starters, you could re-clone the protein with e.g. just a His tag or > move the tag to another end, or put some distance between the end of TEV > site and the protein; or perhaps use no tag at all -- or a different one? > > Is it possible that the tag is messing you up - yes. Is it 'probable' - I > can't say that I know because I've crystallized literally dozens of proteins > with His-tags attached, and more than a few with His-tag and cleavage site. > I prefer a plain His-tag or a cleaved one, to be sure - but I am not quick > to blame the tag (since there are so many other possible things to blame). > > Based on the behavior of your protein after cleavage, it may be that you > have oligomer(s) forming in solution such that cleaved and uncleaved > proteins do not segregate. You may wish to explore other kinds of > chromatographic separation e.g. ion exchange of HIC - they may or may not > work out. You can also consider cleaving your protein at lower > concentration, in the presence of detergents or polyols, etc. > > Cheers, > > Artem > > On Thu, Apr 7, 2011 at 9:37 PM, 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 >> > >
