Hi Raji, Thrombin is a rather good protease and behaves well in a large set of different detergents ( there is a paper by Michael Wiener that describes the relative efficiencies of several usual proteases, amongst those thrombin is inlcuded, used routinely for cleavage of membrane protein fusions in detergents. This is a rather extensive survey henceforth it is very informative.
The variable detergent sensitivity of proteases that are utilized for recombinant protein affinity tag removal James M. Vergis 1, Michael C. Wiener in Protein Expression and Purification 78 (2011) 139–142 thrombin tends to be sensitive to reducing agents so I would stay away from DTT and TCEP , b-mercapto is acceptable in reasonable amounts. We cut in salt concentrations as high as 500 mM (NaCl) at pH 7.5-8.5 with 5-10% glycerol around no chelating agents should be present and imidazole in our hands tends to be an inhibitors (probably because it has some chelating/complexing activity). we have good cleavage in DDM , OG and somehow more difficulties in foscholines but it still cuts reasonnably well given the cost of the enzyme and the targets, the most crucial parameter is Protease/target ratio and incubation time and temp. you can do trials on small scale digests in PCR tubes at different temperatures. we usually cut at 4 or room temp. I hope this helps, Best regards, -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
