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Electrophoresis 2010, 31, 35733579

Ewelina Fic1
Sylwia Kedracka-Krok1
Urszula Jankowska1
Artur Pirog1
Marta DziedzickaWasylewska1,2

Research Article

Comparison of protein precipitation


methods for various rat brain structures
prior to proteomic analysis

Received April 6, 2010


Revised August 4, 2010
Accepted August 12, 2010

Sample preparation is a fundamental step in proteomic methodologies. The quality of the


results from a proteomic experiment is dependent on the nature of the sample and the
properties of the proteins. In this study, various pre-treatment methods were compared by
proteomic analysis; we analysed various rat brain structures after chloroform/methanol,
acetone, TCA/acetone and TCA protein precipitation procedures. The protein content of the
supernatant was also examined by 2-DE. We found that for four of the rat brain structures,
precipitation with chloroform/methanol and acetone delivered the highest protein recovery
for top-down proteomic analysis; however, TCA precipitation resulted in good protein
separation and the highest number of protein spots in 2-DE. Moreover, TCA precipitation
also gave high efficiency of protein recovery if prior sonication procedure was performed.

Department of Physical
Biochemistry, Faculty of
Biochemistry, Biophysics and
Biotechnology, Jagiellonian
University, Cracow, Poland
2
Department of Pharmacology,
Institute of Pharmacology Polish
Academy of Sciences, Cracow,
Poland

Keywords:
IEF / Protein precipitation / Proteomics / Rat brain / SDS-PAGE
DOI 10.1002/elps.201000197

1 Introduction
The term proteomics refers to the analysis of all proteins in
a whole cell, including the description of co- and posttranslationally modified proteins and alternatively spliced
protein variants [1]. Proteomic methodologies have made a
great deal of progress in recent years with the development of
high-resolution 2-DE for protein separation and profiling,
high-sensitivity MS for determination of the molecular weight
of peptides which result from enzymatic cleavage of proteins,
and sequencing of secondary ions, and bioinformatic
approaches to protein identification using software databases.
Sample preparation is crucial for conducting reliable
proteomic analysis [2, 3]. Samples should have a highprotein concentration and be free of salt and other interfering components, such as detergents, nucleic acids, lipids,
etc [4, 5]. Precipitation is widely used for processing of
biological molecules such as proteins [6]. This procedure is
used to concentrate and fractionate the target molecule from
various contaminants. For example, in the biotechnology
industry, protein precipitation is used to eliminate
contaminants commonly contained in blood [7].
Sample preparation depends on the origin of the cells or
the tissue. The first step is usually homogenisation or
sonication followed by protein precipitation and solubilisa-

tion in a suitable buffer. Chloroform, methanol, acetone and


TCA are commonly used as protein precipitating reagents.
In this study, we investigated the efficiency of various
methods for protein precipitation using homogenates
derived from different rat brain structures. The rat brain is a
common model for studying the mechanism of action of
compounds that are used to treat human psychiatric and
neurological disorders [8]. Due to the complexity of brain
tissue, optimisation of protein precipitation methods is
crucial for the analysis of brain proteins. Proteomic studies
can assist in the identification of molecular biomarkers of
diseases and the elucidation of the mechanisms of drug
action. The results obtained in this study may facilitate the
choice of the most optimal methods for the study of
alterations in the rat brain proteome after various behavioural and/or pharmacological treatments. The efficiency
and specificity of contaminant removal were monitored by
the Bradford assay, 1-D SDS-PAGE [9] and 2-D SDS-PAGE
[10] before proteomic experiments were performed.
We also analysed the supernatant (supernatants are
usually removed after protein precipitation) in order to
determine the types of proteins that were lost during sample
preparation; precipitation followed by re-solubilisation in
sample solution rarely gives a 100% yield.

2 Materials and methods


Correspondence: Dr. Ewelina Fic, Department of Physical
Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Cracow,
Poland
E-mail: ewelina.fic@uj.edu.pl
Fax: 148-12-664-69-02

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2.1 Tissues
The efficiency of various methods of protein precipitation
was investigated using four rat brain structures:
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Electrophoresis 2010, 31, 35733579

E. Fic et al.

hippocampus, amygdala, frontal cortex and striatum. The


animals (Wistar rats) were decapitated, their brains were
rapidly dissected and the brain structures of interest were
extracted. The brain tissues were immediately frozen and
stored at 801C until further analysis.

and the mixture was vortexed. One volume of 20% TCA was
then added, and the mixture was vortexed followed by
incubation for 1 h at 201C. After that, the samples were
centrifuged at 15 000  g for 15 min at 41C, 0.5 mL ice-cold
acetone containing 20 mM DTT was added and the mixture
was spun again at 15 000  g for 15 min at 41C. Finally, the
supernatant was removed and the pellet was air dried.

2.2 Sample preparation


2.2.1.4 10% TCA precipitation
Each structure was suspended in 0.5 mL of ice-cold lysis
buffer (7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris, pH
8.5, 0.002% bromophenol blue, 65 mM DTT, 1 mM EDTA
and 1 mM PMSF) and homogenised on ice in three stages:
10, 10 and 5 s (homogeniser Miccra D-1, mode A, Carl Roth
GmbH, Karlsruhe, Germany). The tip of the homogeniser
was rinsed with 1.5 mL of lysis buffer. The homogenate was
then centrifuged at 55 000  g for 1 h at 41C. The resulting
supernatant was transferred to a new Eppendorf tube, and
the protein concentration was determined by the Bradford
assay. The samples were frozen at 201C. For subsequent
experiments, 50 mL of the supernatant was taken.

Experiments were performed at 41C. One volume of 20%


TCA was mixed with one volume of protein sample, and the
mixture was vortexed. After 1 h of incubation at 201C, the
sample was spun at 15 000  g for 15 min at 41C, and the
supernatant was removed. Then, 0.5 mL of ice-cold acetone
containing 20 mM DTT was added, and the mixture was
centrifuged at 13 000  g for 15 min at 41C. The supernatant
was discarded, and the pellet was air dried.

2.3 Analysis of supernatants after protein


precipitation

2.2.1 Protein precipitation


Four different precipitation procedures were used: a mixture
of chloroform and methanol, acetone, a mixture of TCA and
acetone or TCA.
2.2.1.1 Chloroform/methanol precipitation
Experiments were performed at room temperature. Four
volumes of methanol were added to one volume of the
protein sample, and the mixture was vortexed. One volume
of chloroform was then added, and the mixture was
vortexed. The sample was centrifuged at 10 000  g for
5 min and the aqueous methanol layer was removed from
the top of the sample. The proteins remained at the phase
boundary between the aqueous methanol layer and the
chloroform layer. Four volumes of methanol were added,
and the mixture was vortexed. The sample was spun at
10 000  g for 15 min. The supernatant was removed without disturbing the pellet, and the pellet was air dried.
2.2.1.2 Acetone precipitation
Experiments were performed at 41C. Four volumes of icecold acetone containing 20 mM DTT were added to one
volume of protein sample. The mixture was vortexed and
incubated at
201C for 1 h. This was followed by
centrifugation at 10 000  g for 15 min at 41C. The supernatant was discarded and the pellet was air dried.
2.2.1.3 10% TCA/acetone precipitation
Experiments were performed at 41C. Eight volumes of icecold acetone were mixed with one volume of protein sample,

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

After precipitation with organic solvents (i.e. chloroform/


methanol, acetone and TCA/acetone), the supernatant was
dried with nitrogen. Then dried supernatant and supernatant after the TCA precipitation were diluted with purified
water (Millipore, Billerica, MA, USA). Vivaspin columns
(500, cut-off 10 000MWCO, Sartorius, Goettingen,
Germany) were used to remove contaminants, such as salts
and lipids, which were always present in high abundance in
the supernatant, and to concentrate the samples. During
this procedure, the samples were diluted with water several
times. The samples were then suspended in re-hydration
buffer A (7 M urea, 2 M thiourea, 2% CHAPS and 0.002%
bromophenol blue). The samples were precipitated with
TCA and acetone/TCA followed by sonication (ultrasonic
processor UP50 H, Hielscher Ultrasonics GmbH, Teltow,
Germany) to improve protein yield.

2.4 1-D SDS-PAGE


Protein pellets were re-suspended in 50 mL of re-hydration
buffer A (Section 2.3). 1-D SDS-PAGE was performed
according to [9] with a 4% stacking gel and a 12% resolving
gel (Mini-Protean 3, Bio-Rad, Hercules, CA, USA). Gels
were stained by silver nitrate according to [11] with minor
modification.
2.5 2-D SDS-PAGE
IPG buffer (0.5%; GE Healthcare, Uppsala, Sweden) and
20 mM DTT were added to the proteins re-suspended in
re-hydration buffer A (Section 2.3) before the first step of the
2-DE. Hippocampus samples containing 25 mg of total

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Electrophoresis 2010, 31, 35733579

protein were applied to an immobilised pH 310 nonlinear


gradient with 7-cm strips (GE Healthcare). Rehydration
(14 h) was completed using a one-step procedure. IEF was
conducted on an Ettan IPGphor 3 IEF System (GE
Healthcare) at 201C with a current limit of 50 mA per strip.
An IEF program was applied that involved pre-focusing
(200 V for 5 h) followed by four steps: a gradient to 300 V for
0.5 h, a gradient to 1000 V for 0.5 h, a gradient to 5000 V for
1.5 h and 5000 V for 1 h. During pre-focusing, the electrode
wicks were used to improve disposal of excess water, salts
and proteins with pI values outside the pH range of the IPG
strips. Small electrode wicks immersed in miliQ-H2O
(Millipore) were placed at the anode and cathode ends of
the IPG strips just beneath the electrodes. The wicks were
exchanged three times. In order to investigate the influence
of electrode wicks on the quality of protein separation, a
comparative study was conducted.
Prior to conducting SDS-PAGE in the second dimension, the IPG strips were equilibrated according to [10].
After the first dimension separation, the proteins were
separated on Mini-Protean 3 using 12% polyacrylamide gels.
The gels were stained using silver nitrate according to [11]
with minor modifications. The 2-DE analysis of protein
pellets and supernatants was performed in triplicate.
The gels were analysed using ImageMaster 2D
Platinum v6.0 (GE Healthcare) software.

2.6 Determination of protein concentration


The protein concentration of the re-solubilised samples was
determined in triplicate using the Bradford assay. The
efficiency of precipitation was determined as a ratio of
the protein concentration before and after precipitation.
The results presented are an average of at least three
experiments.
The Bradford assay was also used to determine the
protein content after the supernatant analysis.

3 Results and discussion


Proper sample preparation is crucial in order to obtain reliable,
reproducible and significant data, particularly in comparative
proteomic studies where minor differences between experimental and control groups are often meaningful [12]. In this
study, we compared preparations of rat brain samples using a
variety of applications for proteomic technology in
neuroscience. These methodologies are useful for identifying
changes in brain protein expression under different experimental or disease conditions, profiling protein modifications
(e.g. phosphorylation) and mapping proteinprotein interactions in animal models of various diseases.
The efficiency of the pre-treatment methods for protein
precipitation was studied using various rat brain structures.
Figure 1 shows a comparison of the 1-DE separation results.
Electrophoretic images representing the protein profiles of
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Proteomics and 2-DE

3575

each brain structure were very similar. The greatest protein


recoveries (Fig. 2 and Table 1) were achieved with the
chloroform/methanol and acetone precipitation methods.
Precipitation of proteins from the amygdala, hippocampus
and striatum with chloroform/methanol gave a protein yield
greater than 70%. For the frontal cortex, the recovery was
surprisingly low; only 30% of the initial protein content was
recovered. This is probably due to a larger concentration of
hydrophobic proteins in the frontal cortex [13]. Hydrophobic
proteins are relatively difficult to dissolve in aqueous solutions. The use of acetone resulted in slightly less than 70%
recovery of the proteins from the amygdala, hippocampus
and striatum. However, for proteins from the frontal cortex,
the recovery was greater than 50%; therefore, precipitation
using acetone produced better results than those obtained
using the chloroform/methanol mixture (Fig. 2). The
advantage of acetone precipitation is that it is a very feasible
procedure, but it requires a large volume of organic solvent.
Precipitation with either TCA/acetone or TCA led to
approximately twofold lower recovery compared with acetone precipitation. Especially, in case of TCA precipitation,
protein loss was probably due to incomplete solubilisation of
the pellets and the acetone wash step. A portion of the pellet
remained insoluble despite the use of additional re-hydration buffer. It is important to emphasise that the recovery of
proteins was strongly dependent on pellet solubilisation.
The solubilisation of protein pellets after precipitation with
methanol/chloroform took 3060 min, and with acetone and
TCA, it took 90120 min. With acetone/TCA, solubilisation
required approximately 180 min. Finally, we obtained a clear
solution of protein in the re-hydration buffer. A clear solution was observed, but it was not further analysed. There are
a few methods that have been shown to increase protein
recovery, such as sample sonication after TCA precipitation
[14]; however, the insertion of the sonicator tip affects
protein recovery since proteins may coat the tip. The appli`ge,
cation of the bioruptor UCD-200 TM (Diagenode, Lie
Belgium) (15 min, 320 W), which enabled to work in closed
tubes placed in ice bath, caused significant increase of
protein recovery after TCA precipitation (Fig. 2 and Table 1).
2-DE images obtained for hippocampal proteins that were
precipitated using each of the four various methods described
here are shown in Fig. 3. There was no significant difference
in the final composition of proteins, and the number of
identified spots was very similar (approximately, 650 spots per
gel; Table 1). However, detailed analysis revealed that there
were few differences between the various precipitation methods. The spots from the samples without precipitation
(Fig. 3A) were fuzzy and streaky. Precipitation with TCA/
acetone, as well as TCA precipitation, gave better IEF than the
other precipitation methods (Fig. 3B and C). This effect was
especially noticeable for basic proteins and small acidic
proteins. The presence of TCA improved the enrichment of
alkaline proteins. Streaks in the alkaline range of the pH
gradient were common artefacts in the 2-D images. However,
a greater amount of alkaline proteins made the spots more
visible and detectable despite the presence of streaks.
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Electrophoresis 2010, 31, 35733579

E. Fic et al.

Figure 1. 1-D SDS-PAGE electrophoresis. Analysis of proteins from


the homogenate of four different
brain structures after various precipitation methods. Lane 1, homogenate; lane 2, chloroform/methanol;
lane 3, acetone; lane 4, TCA/acetone
and lane 5, TCA. The quantity of the
proteins applied to each lane was
approximately 0.75 mg. Gels were
stained with silver nitrate, and each
experiment was performed in triplicate.

100
Amygdala

90

Precipitation Efficiency %

Frontal Cortex

80

Hippocampus

70

Striatum

60
50
40
30
20
10
0
chloroform/methanol

acetone

TCA/acetone

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

TCA

TCA (+ sonication)

Figure 2. Comparison of the precipitation efficiency of different methods


for various brain structures. The
values of standard deviation errors
are presented as thin line bars at the
top of each column. Each experiment
was performed in triplicate.

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3577

Table 1. Percentage of hippocampal protein recovery and number of protein spots after various precipitation proceduresa)
Precipitation method

Protein amount before


precipitation (mg)

Protein amount after


precipitation (mg)

Percentage of
recovery (%)

Number of spots

Chloroform/methanol
Acetone
10%TCA/acetone
10%TCA
10%TCA (1sonication)

270.98732.51
270.98732.51
270.98732.51
270.98732.51
324.17741.22

242.77756.95
186.15754.78
89.67714.83
68.21736.72
249.60759.22

88.52711.62
68.70717.81
33.0172.80
24.08711.55
77.00719.69

667
665
563
629
699

a) Values are the mean7standard deviation of five independent experiments. The number of spots was determined using ImageMaster
2D Platinum v6.0 software.

Figure 3. 2-DE IEF followed by SDS-PAGE. Analysis of proteins from rat hippocampus after various precipitation methods.
(A) Homogenate without precipitation, (B) TCA, (C) TCA/acetone, (D and F) chloroform/methanol, (E) and (G) acetone. Images
(AD) represent samples prepared with paper wicks, whereas (F and G) represent samples prepared without paper wicks. During IEF,
small rectangular wicks were placed at the anode and cathode ends of the IPG strips just beneath the electrodes. The wicks absorb
excess water, salts and proteins with pI values that lie outside the pH range of the IPG strip.

In addition, the presence of acetone in the TCA/acetone


mixture may cause more efficient delipidation of lipid-rich
biological material, such as brain tissue [10]. More complete
delipidation improves the accuracy of IEF. In the case of
acetone, the small acidic proteins were rather weakly focused
(Fig. 3E and G). The application of paper wicks significantly
improved the isoelectric separation and focusing of spots as
reflected in the 2-DE images (Fig. 3AE). Image analysis
indicated that paper wicks give a sharper image and reduced
streaking. According to Fountoulakis [15], approximately 70%
of the brain proteins identified from the 2-D gels had theoretical pI values between 5 and 8, and 15% had pI values between
4 and 5. This is reflected in the 2-D gels shown in Fig. 3.
Precipitation methods were recently compared by Jiang
et al. [16]; this study, which was performed using human

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

plasma, reported that TCA and acetone precipitation, as well


as ultrafiltration, yielded higher protein recoveries compared
with chloroform/methanol precipitation. TCA precipitation
was one of the best protocols. However, as we mentioned
above, the precipitation methods strongly depend on the
starting material.
The results of the supernatant analysis by 2-DE are
shown in Fig. 4. The protein concentration in the supernatants was beyond the sensitivity of the Bradford assay.
The quantity of loading on each 2-D gel was comparable and
estimated on the basis of 1-DE (computer densitometry
analysis). Figure 4 shows the type of proteins lost during
each precipitation procedure. The method that yielded the
highest protein recovery was acetone precipitation (as can
be concluded from the comparison of supernatants);

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E. Fic et al.

Electrophoresis 2010, 31, 35733579

Figure 4. 2-DE IEF followed by SDS-PAGE.


Analysis of supernatants from rat hippocampus after protein precipitation using
various methods. (A) TCA, (B) TCA/acetone,
(C) chloroform/methanol and (D) acetone.

however, the worst results were obtained from TCA/acetone


precipitation (many spots are visible in the 2-D gels). As
shown in Fig. 4, precipitation of acidic proteins using the
mixture of chloroform and methanol gave a low efficiency,
whereas TCA was not efficient for precipitation of highmolecular-weight proteins. The amount of proteins lost
during precipitation did not exceed 5%.
Unfortunately, despite the presence of denaturants and
detergents, the solubilisation of precipitated proteins was
often incomplete. This may have yielded imprecise
results. The precipitate recovery often depends on
re-dissolving in a smaller volume followed by centrifugation
or filtration. Sonication significantly improved the solubility
of insoluble parts of supernatants. Better solubility may be
achieved by intense and longer mixing or vortexing;
however, it is crucial to avoid excessive foaming
and temperature increases, which may cause protein
degradation.

4 Concluding remarks
The development of standardised, reproducible methods for
protein isolation from tissues offers great potential for the
discovery and validation of biomarkers.
The comparison of protein content in four supernatants
revealed that the TCA/acetone method produced the lowest
efficiency for protein precipitation. Reduced protein loss
(fewer proteins in supernatants) occurred with the methanol/chloroform and TCA methods. The acetone method
yielded the lowest loss of proteins, as determined by analysis
of the supernatant. The acetone method yielded high
& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

precipitation efficiency in comparison to precipitation with


methanol/chloroform, as determined by the protein
concentration. However, as has been shown by Simpson
and Beynon [17], acetone precipitation can, after proteolysis,
lead to selective modification of peptides, which makes MS
analysis more difficult.
Finally, the chloroform/methanol precipitation method
was regarded as the best precipitation method for top-down
proteomics. The advantages of this method are: a lack of
sample cooling, simplicity of performance and the short
duration of the procedure (about 45 min), which minimises
the risk of protease degradation. However, when time and
cost are important factors, one should use chloroform/
methanol precipitation. It is advisable to keep the sample
preparation as simple as possible.
For 2-DE, TCA precipitation was optimal. This method
is laborious and requires low temperature; however, it
results in good IEF and clear spots. The proper sonication
procedure for complete TCA pellet solubilisation is strongly
recommended. These results are presented for the hippocampus, but the conclusions can be extended to the other rat
brain tissues as well (data not shown).
In summary, it is important that the chosen
protein precipitation method is able to effectively
concentrate samples and eliminate contaminants, but such
methods may also have the disadvantages of causing
irreversible protein denaturation and protein insolubilisation. Therefore, special attention must be paid to the
complete solubilisation of protein pellets; however, precipitation procedures rarely yield 100% recoveries. Careful
sample preparation is necessary for successful proteomic
analysis.
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Electrophoresis 2010, 31, 35733579

This work was supported by grant 1/10/0-PBZ-MNiI-2/2/


1/2005 from the Ministry of Science and Higher Education. The
research was carried out with the equipment purchased thanks to
the financial support of the European Regional Development
Fund in the framework of the Polish Innovation Economy
Operational Program (contract No. POIG.02.01.00-12-167/08,
project Ma"opolska Center of Biotechnology).
The authors have declared no conflict of interest.

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