Hi Wenhua Have you tried using 6-8M urea or guanidinium hydrochloride to denature your protein+threonine, then refold by dialysis into a non-threonine-containing buffer? If you are performing ITC, then i guess you already have an efficient activity assay, thus maybe use that as an indicator of successful refolding....combined with successful crystal formation may give you confidence in a correct, uniform fold too.
If your unfolded protein is His-tagged etc and dialysis isn't totally doing the trick, then perhaps immobilising your protein to a His-column might allow you to physically wash away the contaminating threonine. You can then try an on-column refold, or elute and perform dialysis refolding again. Good luck, Rick Salmon On 26 June 2012 10:08, Wenhua Zhang <[email protected]> wrote: > Dear ALL, > > The threonine is found in the active site of my protein structure from > the crystallization in the absence of any threonine containing chemicals. I > presume that's why there was no signals detected with the ITC experiment in > which I titrate my protein with Threonine. In the in vitro biochemical > assay, threonine is one of the substrates in the reconsitituted system. > So could anyone offer me a practical protocol to remove the threonine > from the protein for further experiment to confirm the binding of Threonine > to my protein by ITC. > > Thank you all. > > Wenhua > > Ph. D student in structural biology > > Paris-Sud XI, France >
