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I don't know the exact reaction your protein is performing, but an alternative would be to try to turnover your threonine substrate by adding the other substrates. The reaction product should more easily leave the active site. You might have to do an additional gelfiltration to get rid of excess cosubstrates. Good luck as well, Herman ________________________________ From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Richard M Salmon Sent: Tuesday, June 26, 2012 1:06 PM To: [log in to unmask] Subject: Re: [ccp4bb] practical protocol to remove cell culture derived Threonine from the protein 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 <[log in to unmask]> 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