Budding yeast homogenization: Reproducible protein extracts with protein function retained

This Application Note was developed with the approval of Eva Paige Syzmanski and Oliver Kerscher, based off their publication in the Journal of Visualized Experiments (2013) doi: 10.3791/50921. 

Saccharomyces cerevisiae has historically been heralded as an excellent model organism in molecular research due to its quick reproduction cycle, large population study abilities, ease of growth, and well documented genome1, 2. Therefore this common baker’s yeast is a familiar organism in many research labs. 

Despite the great research benefits of the organism, many complications arise when working with the yeast due to a robust cell wall3. The yeast cell wall is a highly evolved organelle that is adaptive and responsive to environmental stress. The effectiveness of the strong, but elastic cell wall is highlighted by its preservation across many other yeast species. Although beneficial for the organism, the cell wall proves to be an obstacle for researchers interested in analyzing its molecular components due to its difficult disruption. 

Bead mill homogenization, as performed by the Omni Bead Ruptor Elite bead mill homogenizer, offers a robust and quick method for disrupting yeast cells. Bead mills are particularly attractive because of the automated nature of their operation, eliminating variation caused by human error. Their ability to process a large number of samples at once eliminates sample processing bottlenecks, creating the potential for high throughput studies. The Omni Bead Ruptor Elite bead mill homogenizer provides researchers with a rapid, efficient, and reliable method for disrupting yeast cells in a sterile and controlled manner.

Despite the vigorous nature of bead mill homogenization, downstream molecular analyses methods are unaffected and samples are open to a variety of methods including PAGE, Western blotting, protein functional studies, and nucleic acid purification. Herein we describe the methods used by Szymanski and Kerscher to disrupt yeast cells for western blotting and protein functional studies.

Figure 2. SDS-PAGE stained with Coomassie blue of whole cell extracts demonstrating reproducible protein recoveries.

Figure 3. Western blot of Siz1 shows no protein degradation or shearing.