You shouldn't have clogging up problems in the first place. We don't use Pichia but have the same system and I've used it before at the MPI in Martinsried. The system is designed that there are no parts which break, you only have to maintain it on a regular basis and clean it properly. Do you have a manual or pressure driven controller ? With the manual one, people tend to over tighten the valve and thereby ruining the system as a metal spike hits a metal outlet (in the end you grind a hole into the base and can break the whole system). That's really the only part that can be broken. With the pressure controlled system this user error is eliminated. Take it apart, clean it and reassemble it is my suggestion - did you ever took it apart to clean it up or add the vacuum lubricant ? By the way the Hampton heavy duty vacuum grease works very well for that purpose, needs to be replaced every three to four months.
The only other alternative would be a glass beater and regularly autoclave and clean the glass beads. 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 5:33 PM, Cory Brooks 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
