The Michael Rout Lab at the Rockefeller University in New York, NY, initially contacted Retsch Inc. (Newtown, PA) in 2006 to discuss the possibility of using the Planetary Ball Mill to cryogenically grind yeast cell pellets. The aim of the experiment was to explore the construct of nuclear pore complexes located on the cell walls of yeast cells. The decision to use a planetary ball mill and not a cryogenic ball mill (shown in Figure 1) for this application was mainly based on the fact that it produces very small particle sizes, which were considered an important prerequisite for more in-depth analysis of the yeast cells. Particle sizes in the submicron range promote a high yield for the following protein purification protocol.
Figure 1 – The RETSCH CryoMill is also highly suitable for cryogenic cell disruption.
Since the Planetary Ball Mills are not specially designed for cryogenic grinding, in March of 2007 the Rout Lab worked out a very specific protocol (see below) to achieve high yields (~90%) of lysed yeast cells. To date, the Rout Lab continues to utilize the Planetary Ball Mill for cryogenically grinding yeast cells for continued research on the above-mentioned project as well as other current research projects.Retsch Inc. currently has 10 installations of the Planetary Ball Mill PM 100 (shown in Figure 2) within U.S. universities as a result of the Rout Lab collaboration.
Not only in the U.S. is the Planetary Ball Mill used for the cryogenic disruption of yeast cells, but the University of Toulouse (France) hosts the Laboratory of Eukaryotic Molecular Biology (LBME), which follows the Rout Protocol for this application as well and has thus far obtained very good results.
Figure 2 – Planetary Ball Mill PM 100.
The research at LBME focuses on the genetic control of eukaryotic gene expression in normal and pathological contexts. These are fundamental research projects; the objective is to understand the molecular basis underlying pathologies such as, for example, breast cancer. The research relies on several model systems that range from unicellular to whole organisms, from yeast to mammals, including primary and transformed cell lines.
Cell disruption process
Dr. Célia Plisson-Chastang of LBME operates the PM 100 with a 125-mL grinding jar of stainless steel and 7–11 grinding balls with 20 mm diameter of the same material to disrupt the yeast noodles (see Figure 3). The next step is the chromatographic purification of the protein particles, and after that functional and structural analyses using electron microscopy and image analysis. With the PM 100, approximately 90% of the yeast cells are disrupted (see Figure 3).
Figure 3 - Dr. Célia Plisson-Chastang with the PM 100.
Advantages for cryogenic disruption
Dr. Plisson-Chastang appreciates the benefits of this method, and explained,“RETSCH’s PM 100 allows [us] to process up to 25 g of frozen yeast noodles in one working run. With more than 90% of lysed cells, this method is more efficient compared to lysis protocols performed in a liquid environment. Cells and cell grindates are manipulated at very low temperatures (ranging from –80 °C during storage to –196 °C when cooled in liquid nitrogen) all the time, thus preventing our ribonucleioproteic particles of interest to be damaged by released enzymes, such as proteases and nucleases.”
Kyle James is Vice President of Sales, Retsch Inc., 74 Walker Ln., Newtown, PA 18940, U.S.A.; tel.: 267-757-0351; e-mail: firstname.lastname@example.org.
The Rout Lab Protocol is available at http://lab.rockefeller.edu/rout/pdf/protocols/Cryogenic_Disruption_of_Yeast_Cells_PM100. pdf and www.retsch.com/rout-protocol. In addition to this protocol, the following video provides a visual walk-through of the process: http://lab.rockefeller.edu/rout/media/grinding.html.