Thank you all for your suggestions. I try various methods suggested and hope they work.
Regards Venkat On Wed, Jan 22, 2014 at 11:44 PM, Roger Rowlett <rrowl...@colgate.edu>wrote: > 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: rrowl...@colgate.edu > > > > 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. > > > > -- *Venkatareddy Dadireddy, B1-10,Prof. S. Ramakumar's Lab, Dept. of Physics,IISc, Banglore. Cell: 07259492227*
