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Thrombin cleavage of His-tags seems to work pretty well in our hands, which includes mostly undergraduates for the hands-on work. I would encourage you to closely RTFM (read the friendly manual). Thrombin is not all that specific if used at the wrong (too high) concentration, so under the worst of conditions, it may chew up your protein pretty good. We have generally found that (following directions) a series of test cleavage reactions with the thrombin concentration serially diluted by say 10X is necessary to find the optimal thrombin concentration. Once the correct concentration is found, you can scale up. (We normally scale up to 2 mg protein, which we can do in 2 mL batches.) As the thrombin degrades, the appropriate dilution changes. Maybe 10,000-fold this week, maybe 1,000-fold next month. We have obtained really excellent results using thrombin, but don't do it often. So maybe we are just lucky. _______________________________________ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [log in to unmask] On 1/22/2014 1:46 AM, venkatareddy dadireddy wrote: > Dear All, > > My protein is cloned in pET-15b vector, contains His- tag, thrombin > cleavage site and extra sequence of 20 amino acids from vector. I > crystallized without cleaving extra sequence and never got any crystal > hits/crystals. Once, I tried thrombin reaction and it didn't work. > Here I would like to know how efficient the cleavage by thrombin and > it is kind of you, can provide protocol and buffer system for reaction. > > Thank you > Venkat. > * > > *