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 >>
