I know this seems a bit crazy, but I used to lyse s. cerevisiae by pelleting the cells into a 50mL conical, freezing in liquid nitrogen, and using a power drill with a drill bit fitting the caliber of the tube, all at liquid nitrogen temp through periodic (or constant) re-immersion in nitrogen. It seemed to work really well, and it was nice how no proteolysis could take place because of the temp. It yielded a talc-like powder which, once all nitrogen was gone, was resuspended in whatever buffer. This can also be done for e coli pellets, and might be advisable for really proteolysis-sensitive proteins.
JPK On Tue, Nov 2, 2010 at 4:33 PM, Cory Brooks <cbro...@uvic.ca> wrote: > Hello all; > > I have successfully expressed a membrane protein in Pichia pastoris, > however I am having a difficult time with cell lysis. > I have used a Avestin emulsiflex to lyse them, however I have had many > difficulties with the system clogging up, and parts wearing out with the > high pressures. > > So I am wondering what other people out there use to lyse their Pichia? > In particular we have been considering > a microfluidizer > a Retsch mixer mill > a TS-series cell disruptor (Constans systems) > > Any thoughts on these options, or other systems would be much appreciated! > > Best regards, > Cory > > Cory Brooks, Ph.D. > Postdoctoral Fellow > University of Alberta >
