Yeast

Yeast 2011; 28: 795–798.

Published online 4 October 2011 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/yea.1905
Research Article

An improved method for whole protein extraction

from yeast Saccharomyces cerevisiae
Tingting Zhang, Jie Lei, Hanjiang Yang, Kun Xu, Rui Wang and Zhiying Zhang*
College of Animal Science and Technology, Shaan’xi Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University, YangLing,
Shaan’xi 712100, PR China

*Correspondence to: Abstract

Zhiying Zhang, College of Animal
Science and Technology, Shaan’xi
Key Laboratory of Molecular
Biology for Agriculture, Northwest
A&F University, YangLing, Shaan’xi
712100, PR China.
E-mail: zhangzhy@nwsuaf.edu.cn
A new method for protein extraction from yeast Saccharomyces cerevisiae cells is
described. This method involves the use of LiAc and NaOH to enhance the permeabil-
ity of yeast cell wall prior to protein extraction with SDS-PAGE sample buffer. It was
safe and efficient compared to other methods reported so far in the literature. The
proteins extracted with this new method retained their immunoreactive properties
and are suitable for most applications in molecular biology studies. Copyright ©
2011 John Wiley & Sons, Ltd.

Received: 16 June 2011

Keywords: LiAc; yeast; Saccharomyces cerevisiae; protein extraction
Accepted: 30 August 2011

Introduction was easier, quicker but not necessarily more efficient

The yeast Saccharomyces cerevisiae is one of the
most popularly used eukaryotic model systems for
studying a wide range of biological phenomena at
both the molecular and cellular level, including cell
cycle regulation, DNA repair, signal transduction
and cell polarity control. Thus protein extraction
from S. cerevisiae cells for subsequent studies on
extracted proteins such as western blotting consti-
tutes a major technique in many molecular and cell
biology labs.
Yeast cells are protected by rigid cell walls, which
are hard to break. It has been general practice to use
glass beads, which can physically impair yeast cell
walls and help to release yeast cellular proteins. In
1994, Horvath and Riezman reported a method to
extract proteins from yeast cells using sodium
dodecyl sulphate–polyacrylamide gel electropho-
resis (SDS-PAGE) sample buffer, which contained
0.06 M-Tris-HC1 pH 6.8, 10% (v/v) glycerol, 2%
(w/v) sodium dodecyl sulphate (SDS), 5% (v/v)
2-mercaptoethanol, 0.0025% (w/v) bromophenol
blue (Horvath and Riezman, 1994). This method
than that using glass beads. Alternatively, yeast cell
walls can be weakened under alkaline conditions
such as using NaOH-containing buffers: the major
components of yeast cell walls are polysaccharides,
which constitute up to 90% of total yeast cell wall
mass, and are mainly a- and b-D-glucans with minor
amounts of chitin (Klis et al., 2006). These hardy
complex sugar materials function as frameworks for
the yeast cell wall and maintain its mechanical
strength; however, a-glucan and some forms of
b-glucan are soluble under alkaline conditions. It
was found that pre-treating yeast cells with 0.2 M
NaOH, followed by 3 min of boiling in SDS-PAGE
sample buffer, significantly increased the yield of
extracted proteins (Kushnirov, 2000). Furthermore, a
recent study by Ge et al. showed that ionic liquids with
positive charge could further enhance the efficiency of
protein extraction from yeast cells (Ge et al., 2010).
However, their buffer contained 3-(dimethylamino)-
1-propylaminium formate (DMAPA-FA), in which
3-DMAPA was a volatilized and sensitizing sub-
stance that could potentially cause contact allergy in
humans (Brubaker et al., 1979; Foti et al., 2003).

Copyright © 2011 John Wiley & Sons, Ltd.

796 T. Zhang et al.

Such health concern prompted us to look for a Results and discussion

new yeast whole protein extraction method, which
would be more efficient and safer to use in the
laboratory.
Lithium acetate (LiAc) is a key component in
the solution currently used for yeast transformation
(Gietz et al., 1995; Gietz and Woods, 2001). For
unclear reasons, LiAc can enhance the permeabil-
ity of yeast cell walls, which allows introduction
of DNA plasmids into yeast cells. Unlike
DMAPA-FA, LiAc is safe to use and was found
to be harmless in humans (Eigenmann and Burgi,
1978). Based on this evidence, we hypothesized
that LiAc might be useful for protein extraction
from yeast cells. Here we carried out a study to test
this possibility.
Adding LiAc and NaOH
Based on our previous work, yeast cells were most
susceptible to chemical treatment and usually gave
most protein production when they were harvested
prior to stationary phase, when cell density (OD600)
reached about 1.0. We followed the same procedure
to harvest cells in this study.
We first attempted to mix LiAc and NaOH with
SDS-PAGE sample buffer at different ratios. In all
cases, precipitates were found in mixed solutions.
Then, we decided to treat cells with LiAc, NaOH
and SDS-PAGE sample buffer sequentially. We
either pre-treated cells with 0, 0.5, 1.0 or 2.0 M

Figure 1. Optimization of LiAc and NaOH conditions in strain W303a. (A) Adding LiAc and NaOH. Yeast cells were
pre-treated with LiAc and NaOH sequentially as indicated in the figure, followed by protein extraction with SDS-PAGE
sample buffer. (B) Optimization of NaOH condition. (C) Optimization of LiAc condition. (D) Timing and temperature. In
(B – D): lane 1, protein extracted by standard glass bead method. In all panels: lane 10, ProteinRuler II (Trans, China). Yeast
whole proteins were visualized by Coomassie Blue staining. The relative yield of protein was quantified by using densitometric
analysis (Quantity One 4.62, Bio-Rad) of the gels and values are shown below the lanes

Copyright © 2011 John Wiley & Sons, Ltd. Yeast 2011; 28: 795–798.
DOI: 10.1002/yea
Yeast protein extraction by LiAc/NaOH
of LiAc, followed by centrifuging and resuspen-
sion of cells in 0.4 M NaOH, or pre-treated cells
with 0.4 M NaOH, followed by centrifuge and
resuspension in 0.5, 1.0 or 2.0 M LiAc. Each step
took 10 min at room temperature. Subsequently,
cells were centrifuged and resuspended in 100 mL
SDS-PAGE sample buffer, boiled for 5 min and
centrifuged again to remove cell debris. The
supernatant, which contained extracted whole cell
proteins, was transferred to a fresh tube. 10 mL
supernatant was used for 12% SDS-PAGE
analysis. As shown in Figure 1A, we found that
pre-treated cells with LiAc, followed by NaOH,
gave better results in yeast whole protein extrac-
tion. Interestingly, pre-treating cells with LiAc or
NaOH alone could partially extract yeast proteins
but to a much lesser extent compared to the
LiAc/NaOH method.
NaOH and LiAc conditions
NaOH
Yeast cells were harvested and pre-treated with
2.0 M LiAc. Then, cells were centrifuged and trea-
ted with different concentrations of NaOH. Finally,
yeast whole proteins were extracted with SDS-
PAGE sample buffer and an equal volume of
lysate was loaded onto SDS-PAGE for analysis.
As shown in Figure 1B, we found that 0.4 M
NaOH helped the most to extract proteins from
yeast cells. This was somewhat inconsistent with
the finding by Kushnirov, who showed that cells
pre-treated with 0.2–0.3 M NaOH were most
favourable for protein extraction (Kushnirov,
2000). The reason for such a discrepancy
between these two studies was not clear.
LiAc
Similarly, we tested different concentrations of
LiAc. As shown in Figure 1C, we found that
2.0 M LiAc resulted in the most protein extracted
from yeast cells. However, a further increase of
LiAc concentration did not give any better yield
(data not shown).
To further optimize the conditions for protein
extraction, we pre-treated yeast cells with 2.0 M
LiAc for 3, 5 or 10 min at 4  C (on ice), 20  C
(room temperature) or 30  C, respectively. As
shown in Figure 1D, the efficiency of protein
Copyright © 2011 John Wiley & Sons, Ltd.
797
extraction was similar when cells were pre-treated
with 2.0 M LiAc for 3 or 5 min regardless of the
temperature. However, the efficiency reduced
when cells were pre-treated with LiAc for 10 min
at 20 or 30  C, but remained unchanged when they
were kept on ice.
Standard procedure
Yeast S. cerevisiae cells (1.5 mL in YPD) were
harvested prior to stationary phase (OD600 = 1.0)
by centrifugation.
1. Cells were first pre-treated with 2.0 M LiAc
and then 0.4 M NaOH for 5 min on ice. Each
time cells were centrifuged before replacing
solutions.
2. Cells were resuspended in 100 mL SDS-PAGE
sample buffer and boiled for 5 min. Then, the
cell lysate was centrifuged to clear cellular
debris.
3. The supernatant containing yeast whole pro-
tein extract was transferred to a fresh tube
and stored at 80  C.
Applications
To validate the applications of this new method,
we detected the expression of b-actin in yeast S.
cerevisiae cells by western blotting. Yeast protein
Figure 2. Application of LiAc/NaOH yeast whole protein
extraction method. Upper panel: yeast strains S288C,
Sigma1278b and W303a are compared with different
methods. Lower panel: expression of b-actin (42 kDa) was
analyzed by western blotting. G, glass beads
Yeast 2011; 28: 795–798.
DOI: 10.1002/yea
798
was prepared as described above or by other
methods for comparison. Among all tested strains,
our newly developed LiAc/NaOH method resulted
in higher yield of protein than any other methods
in control (Figure 2, upper panel). In all cases,
we were able to detect better expression of
b-actin by western blotting with this new method
(Figure 2, lower panel). Currently, we are using
this new yeast whole protein extraction method
in most of our studies in the lab.
Conclusion
Here we report an LiAc/NaOH protein extraction
method from yeast S. cerevisiae cells. This new
method was safe and efficient compared to other
methods so far reported in the literature. The
extracted proteins retained normal immunoreactiv-
ities and are suitable for most applications in
molecular biology studies.
Acknowledgements
We thank all members of the Animal Genomic Laboratory
for their technical assistance. This work was supported by
a grant from the National Natural Science Foundation of
Copyright © 2011 John Wiley & Sons, Ltd.
T. Zhang et al.
China (#30870119) and the National High Technology
Research and Development Program of China (863
Program) (#2009AA10Z110).
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DOI: 10.1002/yea