Dear Qiangmin, may be cleavage site is buried and not accessible for the protease? I am not familiar with membrane proteins but you can consider this version. Another question: is your protein N- or C- tagged?
08.04.2013 21:22, Qiangmin Zhang пишет: > 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 > -- Eugene Osipov Junior Research Scientist Laboratory of Enzyme Engineering A.N. Bach Institute of Biochemistry Russian Academy of Sciences Leninsky pr. 33, 119071 Moscow, Russia e-mail: [email protected]
