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.
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