APPLICATION NOTE #20143

Budding Yeast Homogenization:
Reproducible Protein Extracts with Protein Functioned Retained
Eva Paige Syzmanski1, Drexel Neumann2, and Oliver Kerscher1
1

Integrated Science Center, Biology Department, The College of William and Mary;

Figure 2

This Application Note was developed
based with the approval of Eva PaigeSyzmanski 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 modelorganism in molecular research due to its
quick reproduction cycle, large population
study abilities, ease of growth, andwell documented genome1, 2. Therefore
this common baker’s yeast is a familiarorganism in many research labs.

Figure 1 Budding Yeast

Despite the great research benefits of theorganism, many complications arise whenworking with the yeast due to a robust cellwall3. The yeast cell wall is a highly evolved
organelle that is adaptive and responsive
to environmental stress. The effectivenessof the strong, but elastic cell wall ishighlighted by its preservation across
many other yeast species. Although
beneficial for the organism, the cell wall
proves to be an obstacle for researchersinterested in analyzing its molecularcomponents due to its difficult disruption.

cells in a sterile and controlled manner.
Despite the vigorous nature of bead millhomogenization, downstream molecularanalyses methods are unaffected andsamples are open to a variety of methods
including PAGE, Western blotting,protein functional studies, and nucleic
acid purification. Herein we describe themethods used by Szymanski and Kerscher
to disrupt yeast cells for western blotting
and protein functional studies.

Bead mill homogenization offers a robust
and quick method for disrupting yeast
cells. Bead mills are particularly attractive
because of the automated nature of theiroperation, eliminating variation caused byhuman error. Their ability to processa large number of samples at once
eliminates sample processing bottlenecks,
creating the potential for high throughput
studies. Bead mill homogenization
provides researchers with a rapid, efficient,and reliable method for disrupting yeast

Yeast cultures were transformed withplasmids carrying a 6xHIS V5-taggedSUMO ligase Siz1, GST tagged Slx5 and a
HA tagged Siz1∆440. Cells were isolated
by centrifugation and resuspended in
PBS with a protease inhibitor cocktail.
Following resuspension, cells wereprepared for storage or downstream
analysis by high-speed centrifugation andsnap-freezing in liquid nitrogen.

Methods

Frozen pellets were added to 2 mL tubes
containing 0.5 mm glass beads

Figure 3

Figure 2 SDS-PAGE stained with Coomassie ofwhole cell extracts demonstrating reproducible
protein recoveries.
Figure 3 Western Blot of Siz1 shows no protein
degradation or shearing.

and 500 µL of an ice-cold lysis buffer.
Samples were homogenized throughbead beating in a Bead Ruptor 24 at 5.5
m/s for 20 seconds, six times,
with 1 minute incubation on slushy ice
between each run. Homogenates wereclarified by centrifugation then protein
precipitation was performed by additionof TCA. Protein pellets were resuspended
and stored at -80 °C for future use. Protein
samples were separated by SDS-PAGE to
demonstrate the reproducibility of theprotein extractions (figure 2). Western
blotting was then performed using ananti-V5 antibody to demonstrate thepresence of the V5-tagged SUMO ligase
Siz1 in the whole cell lysate (figure 3)
Syzmanski and Kerscher further evaluated
if bead milling would impact a proteins
tertiary structure and thus its function.
continued

Methods (continued)
Slx5 and Siz1∆440 both possess a RING
domain that has been reported tonaturally coordinate with Zn2+ ions.
Both proteins were suspended in either
a non-denaturing or denaturing bufferthen subjected to affinity capture
through immobilized metal affinitychromatography. The captured proteins
were then eluted and separated via
1D-PAGE and detected through western
blotting using either an anti-GST or antiHA antibody.The integrity of the proteins
tertiary structure was demonstrated by
the fact that the protein was detectedunder native conditions but absent underdenaturing conditions (figure 4).

Figure 4
Figure 4 Western Blot for
SIx5 and Siz1∆440 shows
recovery following resin
based purification undernative conditions, but not
denaturing conditions.

Conclusions
Syzmanski and Kerscher’s study
established a foundation of proceduresfrom which future yeast research
can be built upon. In their study theyshow the benefit of using bead millhomogenization as a method to efficiently
disrupt yeast in a reproducible andconservative manner. This technology
provides researchers with a fast, user
friendly mechanism to perform highthroughput studies, eliminating sample
prep bottlenecking.

References
1.

Botstein D, Chervitz SA, & Cherry JM.
(1997). Yeast as a Model Organism.
Science. 277(5330): 1259 – 1260.

2.

Ashburner et al. (2000). Gene
Ontology: tool for the unification ofbiology. Nature Genetics. 25: 25 – 29.

3.

Klis FM, Boorsma A, & De Groot PWJ.
(2006). Cell wall construction in
Saccharomyces cerevisiae. Yeast. 23:185 – 202.

Fisher Scientific Bead Mill 24
Catalog No. 15340163