Hi Venkat, For any protease digestion my best liked method is to try digest while the protein is bound to the column. make sure you know your resin dead volume and incubate in the protease buffer overnight. Just after adding enough volume try to rotate (gently, ideally on a roller bottle) for an half an hour or so. before leaving the column upright in cold for overnight incubation. Collect the elute and concentrate before running the gel. Make sure you run a before and after digestion resin samples. Load resin samples equally diluted with your eluted samples to assess the efficiency.
padayatti On Tue, Jan 21, 2014 at 10:46 PM, venkatareddy dadireddy < [email protected]> 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. > > > -- P
