I tried the mortar procedure, and found it took forever, with little chips of thawing yeast inevitably hopping out and sticking wherever, since everything was so brittle, even though I had extruded the pre-frozen pellet through a syringe into liq N2 to make yeast-pellet-noodles first. In contrast, the drill procedure actually was not messy at all, since mostly the stuff was powdered and contained in the tube, so I would drill, empty, drill, empty, etc. I used cryogloves for holding the tube, and the spade-type drill bit was ~$5 at Home Depot.
Jacob On Tue, Nov 2, 2010 at 5:29 PM, Jürgen Bosch <jubo...@jhsph.edu> wrote: > Yes that's how it's conventionally done if you are working with plants. > Instead of a drill (what did safety inspection said to that, did you have > proper precautions in place etc.) they use a mortar cooled in LN2 and also > containing LN2 within the mortar and a suitable grinder plus cryo gloves > (for you). > Then you keep grinding until you have a nice pesto. Thinking about this, we > should do that for smaller amounts instead of diluting the cells so that > they can be run through the Emulsiflex ... Need to go to my freezer and pull > out a pellet and grind them up. > Jürgen > - > Jürgen Bosch > Johns Hopkins Bloomberg School of Public Health > Department of Biochemistry & Molecular Biology > Johns Hopkins Malaria Research Institute > 615 North Wolfe Street, W8708 > Baltimore, MD 21205 > Phone: +1-410-614-4742 > Lab: +1-410-614-4894 > Fax: +1-410-955-3655 > http://web.mac.com/bosch_lab/ > On Nov 2, 2010, at 6:19 PM, Jacob Keller wrote: > > 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 > > >
