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Dear Wenhua, in extension to the post from Herman, you could try to remove the threonine with a separate enzyme, for instance threonine oxidase or amino acid deaminase. Their reaction products probably also bind less tightly. The question is, how fast the release of threonine from your protein is. greetings Gottfried Am Dienstag, dem 26-06-2012 um 11:08 schrieb Wenhua Zhang: 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