GST-purification multiple band problem - GST-purification (Nov/14/2006 )
I'm trying to purify a soluble GST-tagged protein (82kDa) and am having the problem of eluting multiple bands after column purification. I have tried expressing in various bacterial strains ie. BL21, BL21(DE3), Rosetta Blue as well as lowering the temperature for expression to 18 degrees overnight. I have also tried all types of solbule purification methods to help with degradation and non-specific protein sysnthsis and still no luck. Any suggestions would be greatly appreciated. So far my current protocol goes as follows:
- Transform purified plasmid DNA into BL21 (DE3), grow overnight starter culture from colonies
- Diltute stater 1:100 and obtain OD at 0.6-0.8
- Express overnight at 18 degrees using 0.1mM IPTG
- Solubilise bacterial pellet using sonication in TBS with 1% Triton X100, protease inhibitors
- Filter sample and load onto a GSTrap 1ml column
- Elute using 10mM reduced glutathione in TBS
EDTA, PMSF, DTT, lysozyme during solublisation and even batch purifcation using GSH beads have all been trialed and still no difference.
Any help please..
i replied to this earlier at another place
was it the same post??
try more variations of temperature and time fo r growth after induction and IPTG concentrations
check the manual trouble shooting section