This paper compares the activity of various proteases (Prescission is GST tagged HRV 3C) in detergents; PP should be active in DDM.
Vergis, J. M., & Wiener, M. C. (2011). The variable detergent sensitivity of proteases that are utilized for recombinant protein affinity tag removal. Protein Expression and Purification, 78(2), 139–142. doi:10.1016/j.pep.2011.04.011 How old is your prescission stock – we had had some issues with (quite old) protease not working as well as fresh. Alice -- Alice Dawson WNH Lab Division of Biological Chemistry and Drug Discovery College of Life Sciences University of Dundee 01382 385744 From: Qiangmin Zhang <[email protected]<mailto:[email protected]>> Reply-To: Qiangmin Zhang <[email protected]<mailto:[email protected]>> Date: Monday, 8 April 2013 18:22 To: "[email protected]<mailto:[email protected]>" <[email protected]<mailto:[email protected]>> Subject: [ccp4bb] Anyone has experience with digesting membrane protein by precession protease Hello everybody, I just purified a membrane protein tagged with GFP, which has a cleavage site of precession protease. And I got a problem with removing the GFP tag by precession protease (1mM DTT and 1 mM EDTA were included in the buffer). It can not cut my protein. I have already tried to digest it in different detergent like DDM and C12E8 (also different concentration for detergent like lowering the detergent to 1.1 x cmc since a recent science paper lowered the detergent to this level and got it worked). I know this depends on the different proteins. I am wondering if anyone has this experience in digesting membrane protein by precession protease. Any suggestions are appreciated. Otherwise I might have to go back to just his-tag if there is no trick for that. Thank you so much in advance. All the best Qiangmin Zhang The University of Dundee is a registered Scottish Charity, No: SC015096
