ACTA AGRONOMICA SINICA

Volume 36, Issue 4, April 2010
Online English edition of the Chinese language journal
Cite this article as: Acta Agron Sin, 2010, 36(4): 612–619.

RESEARCH PAPER

Construction of Two-Dimensional Polyacrylamide Gel
Electrophoresis System for Proteins in Brassica napus
GAN Lu1,2, LI Dian-Rong1,3, ZANG Xin4, FU Chun-Hua1,2, YU Long-Jiang1,2, and LI Mao-Teng1,2,*
1

Institute of Resource Biology and Biotechnology, College of Life Science and Technology, Huazhong University of Science and Technology,
Wuhan 430074, China

2

Key Laboratory of Molecular Biophysics, Ministry of Education, Huazhong University of Science and Technology, Wuhan 430074, China

3

Hybrid Rapeseed Research Center of Shaanxi Province, Dali 715105, China

4

Bioengineering Department of Zhengzhou University, Zhengzhou 450001, China

Abstract: The purpose of this study was to establish a suitable 2-dimensional polyacrylamide gel electrophoresis (2-DE) system for
proteomics analysis in Brassica napus. Proteins of rapeseed variety 08127 were separated in gel brands (pH 4–7) using trichloroacetic
extraction method under the improved isoelectric focusing (IEF) procedure. A clean 2-DE map was obtained, indicating that the system
was effective and suitable for B. napus. This system was validated using young seedling, leaves, and stems from other varieties, and the
results were similar to that of variety 08127. The proteomics profiling of seedlings in different ages of B. napus was then analyzed on the
basis of this system. In the stems of 30-day-age seedlings, 20 up-regulated and 51 down-regulated proteins were found with differential
expression more than 2 folds compared to their corresponding proteins in 15-day-age stems. A few differential protein spots were
identified using mass spectrometry method, indicating the effectiveness of the 2-DE polyacrylamide gel electrophoresis system
established in this study.
Keywords: Brassica napus; protein; two-dimensional polyacrylamide gel electrophoresis

Two-dimensional polyacrylamide gel electrophoresis (2-DE)
technique is able to separate more than 1800 protein spots in a
single gel [1]. Thus, it is suitable for analyzing thousands of
gene expression products simultaneously, and has become the
main and preferred technique in proteomics research [2, 3],
especially in discovery of specific function of proteins and
genes [4]. The 2-DE technique, which was firstly established
by O’Farrell et al. [5] in 1975, separates proteins on the basis
of the variations of isoelectric point and protein molecule
weight of proteins and performs isoelectric focusing (IEF) at
both horizontal and vertical directions in sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
However, it has not been widely accepted by researchers until
Bjellqvist et al. [6] invented immobilized pH gradients in 1982
to eliminate the cathode drift and poor reproducibility of
traditional IEF [7]. Continuous improvement of this technique
ensures its better resolution and repeatability in proteomics

researches.
To date, the 2-DE technique has been used proteomics
studies of numerous plant species, such as Arabidopsis
thaliana [8–10], rice (Oryza sativa L.) [11, 12], soybean [Glycine
max (L.) Merr.] [13], tobacco (Nicotiana tabacum L.) [14], and
tomato (Lycopersicon esculentum Mill.) [15]. Most reports are in
relation to proteomics of physiology, genetic diversity, mutants,
and development in plants [16]. In B. napus, Luis et al. [17]
investigated the differences of proteomics and gene
expression in seedlings when exposed to low temperature.
Mihr et al. [18] compared the protein expression profiles
between the cytoplasmic male sterile B. napus and the wild
type, and found a remarkable difference. Hajduch et al. [19]
found 729 differentially expressed protein spots during seed
filling of B. napus. Agrawa et al. [20] investigated the difference
of protein expression between soybean and B. napus in the
process of seed maturing. Besides, Sheoran et al. [21] studied

Received: 8 September 2009; Accepted: 24 December 2009.
* Corresponding author. E-mail: limaoteng426@163.com or limaoteng426@mail.hust.edu.cn
Copyright © 2010, Crop Science Society of China and Institute of Crop Sciences, Chinese Academy of Agricultural Sciences. Published by Elsevier BV. All rights reserved.
Chinese edition available online at http://www.chinacrops.org/zwxb/
DOI: 10.1016/S1875-2780(09)60046-8

GAN Lu et al. / Acta Agronomica Sinica, 2010, 36(4): 612–619

the dynamics of protein expression during the germination of
B. napus pollens. However, the proteomics in B. napus has
been rarely investigated in China.
The purpose of this study was to establish an applicable
method for protein extraction and IEF procedure in B. napus.
The system was validated with B. napus seedlings at different
developmental stages, and a few valuable protein spots were
obtained. The protocol constructed may contribute to the
proteomics research of B. napus.

1

Materials and methods

1

Plant materials

Seeds of oilseed rape cultivar 08127 were grown in plastic
box filled with the mixture of vermiculite and perlite (V/V 2:1).
Plants were watered with nutrient solution every 2 d. Leaves
and stems of 15- or 30-day-age seedlings were sampled for
different experiments. The nutrient solution contained 2 mmol
L–1 KNO3, 3 mmol L–1 CaCl2, 1 mmol L–1 K2SO4, 0.5 mmol
L–1 MgSO4, 0.4 mmol L–1 KH2PO4, 0.15 mmol L–1 K2HPO4,
0.2 mmol L–1 Fe-Na-EDTA, 14 mmol L–1 H3BO3, 5 mmol L–1
MnSO4, 3 mmol L–1 ZnSO4, 0.7 mmol L–1 (NH4)6Mo7O24, 0.7
mmol L–1 CuSO4, and 0.1 mmol L–1 CoCl2 [22].
1.2

Protein extraction

Proteins were extracted from seedling leaves or stems using
the trichloroacetic acid (TCA)/acetone method described by
Jacobs et al. [23] and the Tris-HCl method described by Gu et
al. [24] with minor modifications, respectively.
In the TCA/acetone method, 1 g of leaves or stems was
ground into fine powders in liquid nitrogen after washing with
ultrapure water. The powders were added with 10 volumes of
precooled (–20qC) acetone, which contained 10% TCA and
0.07% dithiothreitol (DTT), and the mixture was incubated at
–20qC for 1 h and centrifuged with 12,000u g for 30 min at
4qC. The precipitate was then incubated in 10 volumes of
acetone (containing 0.07% DTT) and centrifuged thrice as
described above. Finally, the precipitation, after air-drying at
room temperature, was resolved into 500 μL of lysis buffer
containing 7 mol L–1 urea, 2 mol L–1 of thiourea, 4% 3-[(3chloramidopropyl) dimethylammonio]-1-propanesulfonate
(CHAPS), 1 mmol L1 of phenylmethanesulfonyl fluoride
(PMSF), 50 mmol L–1 of DTT, 0.5% Triton X-100, and 0.5%
ampholine. The solution was incubated at 25qC for 1 h with
gentle mixing and then centrifuged at 12,000u g for 20 min.
Supernatant was collected into fresh tubes and stored at –80qC
in aliquots.
In the Tris-HCl method, 1 g of plant powders were washed
with ultrapure water and extracted in the buffer (65 mmol L–1
Tris-HCl, pH 6.8, 0.5% sodium dodecyl sulfate (SDS), 10%
glycerol, and 5% ȕ-mercaptoethanol) under shaking for 1 h at
4°C. The supernatant was collected after centrifugation at

12,000× g at 4°C for 30 min. Proteins were precipitated at
–20°C for 1 h with 10× precooled acetone containing 0.07%
DTT. The precipitate was then collected by centrifugation at
12,000× g for 30 min, washed with 10 mL of precooled
acetone containing 0.07% DTT at –20°C for 1 h, and
centrifuged thrice as described above. The final precipitate
was air-dried, dissolved in the rehydration buffer (containing
7 mol L–1 urea, 2 mol L–1 thiourea, 4% CHAPS, 65 mmol L–1
DTT, 0.2% ampholine, and 0.001% bromophenol blue), and
stored at –80°C.
The quality of extracted protein was tested using the RC
DC protein assay kit (Bio-Rad Laboratories, Inc., Hercules,
CA, USA) according to the manufacture’s instruction. The
standard protein was the bovine serum albumin.
1.3

Methods for 2-DE and image analysis

IPG strips of 17 cm were actively rehydrated with 300 μL
of rehydration solution for 14 h at 50 V, which was
contained 1 mg of protein. Isoelectric focusing (IEF) was
carried out after adding the extraction solution containing 1
mg of protein and the IEF loading buffer (7 mol L–1 urea, 2
mol L–1 thiourea, 4% CHAPS, 65 mmol L–1 DTT, 0.2%
ampholine, and 0.001% Bromophenol blue) was added to a
final volume of 300 μL. The procedure for IEF is listed in
Table. After IEF separation, the strips were equilibrated
twice for 15 min each using equilibration buffer (6 mmol
Lí1 urea, 0.375 mmol Lí1 Tris-HCl, pH 8.8, 20% glycerol,
and 2% DTT) with 2% DTT and 2.5% iodacetamide (IAA).
The second dimension separation of proteins was performed
in 10% SDS-PAGE gels using Bio-Rad PROTEAN II xi
Cell system (Bio-Rad Laboratories, Inc., Hercules, CA,
USA). The electrophoresis initiated with a constant electric
current of 10 mA for 45 min and was switched to 20–30 mA
until the bromophenol blue frontier reached the bottom of
the gel. The gel was fixed for 1 h in a solution containing
40% methanol and 10% acetic acid, washed for 15 min with
deionized water, and then stained in a staining buffer (0.12%
Coomassie brilliant blue G-250, 10% ammonium sulfate,
10% phosphoric acid, and 20% methanol) for 24 h [25], and
then decolored use ddH2O until the background was clear.

Table Running conditions of isoelectric focusing
Step

Voltage (V)

Voltage increase model

Time

S1

50

Linear

14 h

S2

50

Linear

1h

S3

200

Linear

1h

S4

500

Linear

1h

S5

1000

Linear

1h

S6

10000

Linear

S7

10000

Accelerated

80,000 VHr

S8

500

Accelerated

Any time

VHr: volt-hours.

5h

GAN Lu et al. / Acta Agronomica Sinica, 2010, 36(4): 612–619

1.4

Scanning and analysis of images

Images were obtained using UMAX Powerlook 2100XL
(UMAX, Taipei, China) at a resolution of 300 dpi and 16-bit
grayscale pixel depth, and analyzed using PDquest 8.01
software (Bio-Rad Laboratories, Inc, Hercules, CA, USA). All
spots was normalized to the total density in the gel image and
was expressed in parts per million.
1.5

Mass spectrometry

Mass spectrometry analysis of selected protein spots were
carried out in Shanghai Applied Protein Technology Co. Ltd.
(Shanghai, China) using Matrix-Assisted Laser Desorption/
Ionization Time of Flight Mass Spectrometry (MALDI-TOFMS) method [26]. The protein spots were excised from the gels
with pipette tip or using the Ettan Spot Picker and transferred
into the 1.5 mL tubes separately for in-gel digestion. After
washing twice with Milli-Q water, gel spots were destained in
a 1:1 destaining solution (30 mmol L–1 of potassium
ferrocyanide, 100 mmol L–1 of sodium thiosulfate, and 100
mmol L–1 of ammonium bicarbonate, pH 8.0) for 20 min.
Following washing twice with Milli-Q water, the samples
were dehydrated with acetonitrile vacuum drying. The gel
pieces were rehydrated in 10–20 μL of digestion buffer and
then incubated at 37°C overnight. Peptides were extracted

pH 3

pH

twice with 50 μL of 50% acetonitrile and 5% trifluoroacetic
acid for 15 min each before vacuum dried. Finally, 10 μL of
1% trifluoroacetic acid were added to dissolve the extracted
peptides for sample plating [26].
This peptide solution (2 mL) was mixed with equal volume
of Į-cyano-4-hydroxycinnamic acid (10 mg mL–1) saturated
with 50% acetonitrile in 0.1% trifluoroacetic acid. Samples
were analyzed using a BrukerAutoFlex MALDI-TOF mass
spectrometer (Bruker Daltonics, Billerica, Massachusetts,
USA) with the following parameters: 20 kV of accelerating
voltage, 60–65% of grid voltage, 160 ns of delay extraction
time. Laser shots at 200 per spectrum were used to acquire the
spectra with a mass range from 1000 to 4000 Da. The
MASCOT search engine (http://www.matrixscience.com) and
the NCBI protein database (http://www.ncbi.nlm.nih.gov) were
used to identify the peptide mass fingerprinting (PMF) [26].

2
2.1

Results
Evaluation of protein extraction methods

Compared with the Tric-HCl method, the TCA/acetone
extraction method produced better focus, cleaner background,
and streak-free result in both vertical and horizontal directions
in the 2-DE gels (Fig. 1-a and 1-b). Although more protein

pH 3

pH 10

c

Fig. 1 Two-DE profile of Brassica napus proteins extracted using TCA/acetone and Tris-HCl methods
a: TCA/acetone extraction method; b: Tris-HCl extraction method; c: 2-DE map of B. napus proteins extracted using TCA/acetone method
(separated with 17 cm strips, pH 4–7). Boxed areas in Fig. 1-a are amplified in Fig. 1-c, showing the improved separation of protein spots.

GAN Lu et al. / Acta Agronomica Sinica, 2010, 36(4): 612–619

spots were detected using Tris-HCl extraction method than the
TCA/acetone method, the contamination was obviously in the
2-DE gels that had a negative influence of analysis. Therefore,
the TCA/acetone extraction method is recommended in the
2-DE analysis of B. napus.
In the pH 3–10 IPG stripes from the 2-DE gels using both
extraction methods, the protein spots of B. napus were mainly
distributed in the pH 4–7 IPG strips. To obtain a better
separation result, the proteins extracted using TCA/acetone
method were further separated in 2-DE gels with pH value
ranging from 4 to 7. The result showed an improved
separation with more clear protein spots (Fig. 1-c), and a total
of 382 protein spots were observed.
2.2

Effect of IEF time on 2-DE profile

IEF is a determinative factor on the final result of 2-DE.
According to the manufacturer’s instruction, the recommend
total focusing voltage is 60,000 VHr (volt-hours) after the volt
rising to 10,000 V for the 17 cm IPG strip. However, the total
focusing voltage of 60,000 VHr were not sufficient in for the
2-DE of B. napus, even the total focusing voltage of 70,000

VHr were not sufficient either (Fig. 2-a). Based on our result,
when 1 mg of sample was loaded to the 17 cm IPG strips with
pH values ranging from 4 to 7, the fine 2-DE gel profile with
clear protein spots was obtained under the total focusing
voltage of 80,000 VHr (Fig. 2-b). Using both stem and leaf
samples of B. napus, similar quality of 2-DE profile was
obtained under the total voltage of 80,000 VHr (Fig. 2-c and
2-d), showing that this protocol is applicable for different
tissues of B. napus plant.
2.3 Comparison of 2-DE profiles from seedling samples
with different ages
Proteins from 15- and 30-day old seedlings of B. napus
were extracted using the TCA/acetone method. There were
329 and 372 protein spots according to the 2-DE profiles,
respectively (Fig. 3-a and 3-b), of which 181 protein spots
were differentially expressed. The quantities of differentially
expressed protein spots could be illustrated with 3-dimension
profiles produced by PDquest 8.01 software (Fig. 3-c and 3-d).
Among the 181 differentially expression protein spots, 118
spots were shown in changing expression level more than 1.1

Fig. 2 Effect of total focusing voltage on 2-DE profile with TCA/acetone extraction method
a: 2-DE profile under total focusing voltage of 70,000 VHr; b: 2-DE profile under total focusing voltage of 80,000 VHr, showing better
separation of protein spots (arrows); c and d: 2-DE profiles with stem and leaf samples of B. napus, respectively.

Intensity (%)

GAN Lu et al. / Acta Agronomica Sinica, 2010, 36(4): 612–619

Fig. 3 Two-DE profiles and differentially expressed protein spots in Brassica napus seedlings of 15-day and 30-day ages
a and b: 2-DE profiles of 15-day and 30-day old seedlings, respectively; a’: One of the differential spots (in the circle) that is extracted and
amplified from picture a; c and d: Three-dimension profiles of the selected parts of 2-DE gels; e: Mass spectrementy analysis of proteins in the
a’ region, showing 17 peptide mass fingerprints in this region. The section in a rectangle is amplified in a separated graph coded as e’.

folds, and 71 spots in changing expression level more than 2.0
folds including 20 up-regulated and 51 down-regulated
protein spots. A total of 5 up-regulated and 9 down-regulated
spots were observed with the expression quantity change more
than 5 folds.
To test the feasibility of protein identification using protein
spots from 2-DE gels, several protein spots, such as the spot

shown in Fig. 3-a’, were selected for mass spectrometry
analysis (Fig. 3-e and 3-e’). The protein spot shown in Fig. 3-a’
was separated into 17 peptide mass fingerprints. This protein
was found to be homologous (99.44%) to a transporter in
Arabidopsis thaliana (PATELLIN 1) using the MASCOT
search engine against the NCBI protein database to analyze
the PMF of this spot. This result indicates that the 2-DE

GAN Lu et al. / Acta Agronomica Sinica, 2010, 36(4): 612–619

system established is applicable for protein identification.

3

Discussion

The optimal 2-DE profile is obtained on the basis of several
key steps. Protein extraction is the first and the determinative
step on the final result of 2-DE [27, 28]. Salt, water-soluble
organic matter, and macromolecular-charged groups are
disadvantageous to protein samples for 2-DE, and the
principle of sample extraction is to remove these interfering
substances as much as possible. At present, the most common
methods for extraction of plant protein include acetone
precipitation, TCA/acetone precipitation, and phenol
extraction methanol/ammonium acetate precipitation. Proteins
can be deposited with acetone quickly using the TCA/acetone
extraction method, but lots of carbohydrates that are deposited
together with proteins simultaneously, leading to the bad
quality of 2-DE profile. The phenol extraction methanol/
ammonium acetate extraction method is complex in procedure,
but it can effectively remove organic impurities through
dissolution of different phases. The suitable extraction method
is different subject to plant materials because different
materials may contain different carbohydrates, fats, pigments,
and other substances [29]. Therefore, proper modification of
protein extraction method is of great importance to obtain the
best separation profile of protein spots.
Protein lysis is another important step for sample
preparation. This step aims at ensuring dissolution of protein
as much as possible on the basis of maintaining its biologic
activity [27]. In this study, the proteins of B. napus were
extracted using the TCA/acetone method and treated with
lysis buffer. The extracted protein samples were very clean
with very few nucleic acids and salt, resulting in voltage
increase to 10,000 V during IEF and good 2-DE profiles. This
protocol is universally applicable to different organization of
B. napus, such as seedling, leaf, and stem. These results
indicate that the protocol established in this study is suitable
for 2-DE research in B. napus.
The final 2-DE profile is also affected by IEF. Protein
samples must be focused at high voltage to obtain good 2-DE
gels. One common problem is that the voltage cannot be
raised to the appointed value due to the high content of salt in
the samples, resulting in focusing deficiency during IEF.
Finally, horizontal streaks appear on the 2-DE gel. However,
excessive focusing can also induce horizontal streaks in the
2-DE gel. In this study, several total focusing voltages were
tested and 80,000 VHr is adopted as the optimal parameter for
fine 2-DE gels.
Strip equilibration, which aims at promoting interaction
between protein and SDS, directly affects protein separation
on the second dimension. The equilibration buffer contains
Tris-HCl (pH 8.8), SDS (2%), and high concentrations of urea

(6 mol L–1) and glycerol (20%). DTT (1%) added in the first
step of equilibration is to enhance protein unfolding, and IAA
(2.5%) added in the second step of equilibration is to remove
superfluous DTT so that to prevent tailing in silver staining.
Parts of proteins (5% to 25%) might be lost during the
equilibration, which reduces the resolution of 2-DE gels. No
equilibration or deficient of equilibration may result in texture
phenomenon of high molecular weight protein and the IEF gel
might be glued to SDS-PAGE. Thus, the principle of strips
equilibration time should be sufficient (at least 10 min twice
but no more than 15 min for each time. The protein profiling
of 15- and 30-day-age B. napus seedlings was analyzed using
the protocol established, and 329 and 372 protein spots were
observed including 20 up-regulated and 51 down-regulated
proteins with more than 2 folds. Some differentially expressed
protein spots were successfully identified. Our future study
will focus on the identification and analysis of differential
spots.

4

Conclusions

The TCA/acetone extraction method is suitable for
extracting proteins from different organs/tissue of B. napus
seedlings. This method, together with proper IEF procedure,
is pivotal for obtaining fine 2-DE profiles. Using the 2-DE
system established, proteomics from 15- and 30-day old B.
napus seedlings were compared, and differentially expressed
protein spots were detected. Some of the differential protein
spots were identified using MALDI-TOF MS. The result
indicates that the 2-DE system established is suitable for B.
napus.

Acknowledgments
The study was supported by the National High Technology
Research
and
Development
Program
of
China
(2009AA101105), the Program for New Century Excellent
Talents in University of Ministry of Education, and the fund
of Huazhong University of Science and Technology
(M2009059).

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improvement of two-dimensional electrophoresis analysis of
proteins form berries of Elaeagnus umbellate Thunb. Plant
Physiol Commun, 2009, 45: 695–698 (in Chinese with English
abstract)

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