how to isolate insoluble protein in bacterial induction?
Posted 10 September 2009 - 07:19 AM
I am trying to expressed a piece of my protein of interest in PGEXT4T1 vector (GST tag). I induced the cell at OD 0.6 with IPTG for 3hrs at 30C. However, the protein is insoluble and stayed in the pellet after sonication. I tried to treat the pellet with triton X-100 and load the whole complex and probe with GST and see the protein (a band with the corresponded size). I read in other topics people talking about inducing at lower temperature for a longer period of time. I just wonder if there are other alternatives to move the proteins out of the pellet?
Posted 10 September 2009 - 04:02 PM
Failing that, temperature and IPTG concentration are the standard ways to try to reduce protein going into the insoluble fraction.
As a complete alternative, will your project suffer if you let the protein go insoluble and purify by denaturing it and refolding? Some inclusion bodies have been teased apart by slowly increasing the urea concentration, kind of like an ammonium sulphate precipitation in reverse. St out at 1 M, which is often used to clean other contaminating proteins off the surface of the IB. You'll have to go slowly, adding the urea, incubating, spinning down to see what happens to the pellet and adding more urea as appropriate. Real old-school, bootstrap stuff, but it might work if you don't want to smash the IB into submission with 8M urea.
Posted 14 September 2009 - 08:21 AM
Posted 14 September 2009 - 02:34 PM
Posted 05 October 2009 - 07:17 AM