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Protein extraction/solubilization protocol for

monocot and dicot plant gel-based proteomics

ARTICLE in JOURNAL OF PLANT BIOLOGY · NOVEMBER 2006

Impact Factor: 1.21 · DOI: 10.1007/BF03031120

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Journal of Plant Biology, December 2006, 49(6) : 413-420

PTotein E• Protocol for Monocot and

Dicot Plant GeM-Based Pro eomics
Kyoungwon Cho ~, N[ika [.[nelh Torres2~ Subhashree Subramanyam 3, Saligrama A Deepak 4,
Nagesh Sardesai s, Oksoo Hart ~, Christie E. WH|iams 6' 7, Hideo |sh[i 4,
Hitosh[ Iwahash[ 8, and Randeep Rakwa[ 8.
~Department of Applied Biotechnology, Agricultural Plant Stress Research Center, and Biotechnology Research Institute,
Chonnam National University, Kwangiu 500-757, Korea
2University of Panama, University Re~ional Center of Azuero, Province of Herrera, Panama
3De'partment of Agronom,/, Pur~due University, West Lafayette, IN 47907, USA
4National Institute for Agro-Environmental Sciences, Ibaraki 305-8604, Japan
SDepartment of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA
~Department of Entomology, Purdue University, West Lafayette, IN 47907, USA
7USDA-ARS Crop Production and Pest Control Research Unit, Purdue University, West Lafayette, IN 47907, USA
8Human Stress Signal Research Center, National Institute of Advanced Industrial Science
and Technology WEST, Ibaraki 305-8569, Japan

Sample preparation in plant proteomics is tedious, requiring modifications depending on the type of tissue involved. Here,
we describe a protein extractlon protocoM for both monocotyledonous (monocot) and dicotyledonous (dicot) species, which
significantly improves the soiubilization of total proteins~ For exam#e, we used the primary leaf tissue and seeds from rice, a
cereai crop and genome model system. Total protein was first precipitated with trichRoroacetic acid/acetone extraction buffer
(TCAAEg) and subsequently soiubiNized with a modified O'Farre[] ]ys[s buffer (LB) containing thiourea and tris (LB-TT). Separa-
tion of total leaf proteins by two-dimensional gel eledrophoresis (2-DGE) revealed improved solubillzation, as determined by
an increased number of spots detected with Coomassie brliliant blue (CBB) staining~ In addition, the resolution was better
than when [LBoTTwas used alone for protein extraction~ Seed proteins could be extracted in LB-TT itself without the need for
TCAAEB, which resulted in a highRy [nsolub]e precipitate. Our Cgg-stained 2oD ge[ protein profiles Mso demonstrated the effi-
cacy of this protocol for total protein ext~acfionlsolubilization from the dico~ genome mode] (Arabidopsis), a dicot disease
model (cucumber), and two other important monocot cereal crop models (maize and wheat)~ Moreover, this is the first report
on generating a 2-D gel proteome profile for wheat crown and cucumber leaf t[ssueso Finally, as examples of proteome refer-
ence maps, we obtained silver nitrate-stained, ~arge-format 2-D gels for rice leaf and wheat crown LBoTTso[ubilized proteins,
Keywords:Arabidopsis,cucumber, lysis buffer, maize, rice, wheat

Proteomics, a rapidly progressing discipline in this func- tion, and thus are demanding increased research attention
tional genomics era, is essential not only for determining the in the field of plant sciences. Moreover, because only the
functional components of genes and proteins, but also, and rice genome has been almost completely sequenced (Inter-
more importantly, for addressing the question of post-trans- national Rice Genome Sequencing Project, 2005), this plant
lational modifications because the one-gene/one-protein has become a crucial reference for monocots, and serves as
theory no longer holds true (Pandey and Mann, 2000; Han- a cornerstone for functional genomics in cereal crops
cock, 2004a, b; Agrawal et al., 2005a, b, c; Agrawal and (Agrawal and Rakwal, 2006).
Rakwal, 2006). Two major methodologies have now been Our current study focuses on protein extraction from vari-
identified in proteomics -- two-dimensional gel electro- ous model plants: rice, Arabidopsis (Kim et al., 2005),
phoresis (2-DGE) and mass spectrometry (MS) - also referred maize, wheat, and cucumber. One of our objectives has
to as gel- and non gel-based technologies, respectively been to develop an extraction protocol that can accommo-
(O'Farrell, 1975; Rabilloud, 2002; Yates, 2004; Agrawal date at least all the mode[ plants, and be coupled with the
and Rakwa[, 2006). These two are the basis for current pro- improved extraction and sohbilization of proteins for down-
teomics studies, and are used for separating and identifying stream 2-DGE. Here, we briefly describe the rationale
proteins at the proteome level. Compared with yeast and behind investigating these particular species and stresses at
animal proteomics, that of plants species lags in the amount the proteome level. As with any other "omic technology",
of its research and publications, not because the technology one of the most critical steps in initial experimental design
is lacking, but mostly because the plant genome sequence for proteomics is good sample preparation. Because plant
database is small and sampling complex (Agrawal and Rak- tissues are complex and diverse, protein extraction becomes
wal, 2006). Although Arabidopsis thaliana is an excellent a first and limiting step for subsequent separation and identi-
genome model, rice (Oryza sativa L) and other cereal crops fication by 2-DGE or MS. Despite inherent drawbacks, such
are the most important food sources for the world's popula- as the non-separation of basic and membrane proteins, we
must emphasize that 2-DGE is still one of the most efficient
*Corresponding author; fax +81-29-861-8508 proteomics techniques, providing a visual method for iden-
e-mail rakwal-68@aist.go.jp tifying relative quantitative differences between proteins

413
414 Cho et al. J. Plant Biol. Vol. 49, No. 6, 2006

expressed in a given sample under normal and stress condi- nitrogen before storage at -80~
tions. These proteins can be unambiguously identified using Maize: Maize (Zea mays L. cv. Guarare 8128) plants were
either the classic N-terminal amino acid sequencing approach grown for 16 d in the greenhouse under natural light, at a
or high-throughput MS via matrix-assisted laser desorption/ion- controlled temperature of 25~ and 70% RH. Leaf segments
ization (MALDI)-time of flight (TOF)-MS (MALDI-TOF-MS) and (about 2 cm long) were cut, with clean scissors, from the
liquid chromatography tandem mass spectrometry (LC-MS/ third and fourth leaves of randomly selected seedlings,
MS). Here, our focus is on trichloroacetic acid (TCA)/ace- divided into 100-mg samples, then immediately ground in
tone extraction buffer (TCAAEB), coupled to a modified lysis liquid nitrogen or kept frozen at -80~ until use.
buffer (I_B) for solubilization of precipitated plant proteins in Wheat: Wheat (?riticum aestivum L. em Thell., line 'Iris')
order to have good resolution on 2-DGE. seedlings were grown in 3 x 21 cm Ray Leach Containers
(USA) that were filled by two-thirds with Turface MVP soil
conditioner (Profile Products, USA) for easy root removal.
MATER~IAILSAND METHODS These were then topped with soil as described previously
(Subramanyam et al., 2006). Throughout the experimental
Plant Materia|s and Growing Conditions period, the growth chamber was maintained at 18~ and
under a 24-h photoperiod from light illumination at 250 to
Rice: Seedlings of (9. sativa L. japonica-type cv. Nakdong 300 mol m -2 s-1. The plants were watered as required and
were grown for 14 d under white fluorescent light (wave- fertilized with Peter's fertilizer (WR Grace, USA). Both the
length 390 to 500 nm, 150 tool m -2 s-~, 12-h photoperiod) plant crown (extending from the junction of the root and
at 25~ and 70% relative humidity (RH) (Agrawal and Rak- aerial portion to about 1 cm below the ligule of the first leaf)
wal, 2006). Leaf segments (about 2 cm long) were cut, with and the leaf blade tissue were harvested into liquid nitrogen
clean scissors, from the third and fourth leaves of randomly and stored at -80~ The embryos (50 mg) of wheat (cv.
selected seedlings, divided into 100-rag samples, and imme- 'Norin 60') were carefully excised from dry, mature seeds
diately ground in liquid nitrogen or kept frozen at -80~ using a carbide bur (11/4 size; Emesco, Germany) and scalpel
until use. For the seed, 150-rag samples of freshly de- under a light microscope.
husked, dry, mature seeds from field-grown cv. ~ Cucumber: Cucumber (Cucumis sativus L., cv. 'Shin Suyo
were used as experimental material. TsukemidorP) seedlings were raised in a seeding box con-
Arabidopsis: Seeds of Col-0 (~ were sown on 2 taining cultivation soil (Kureha Chemical, Japan) for easy
x 2.3 cm 2 blocks of glass wool (Minipot; Nittobo, Japan), removal of their roots. After approximately 10 d, the seed-
where they were kept for 2 d at 4~ for vernalization. After- lings were transplanted to 10 • 10 cm plastic pots contain-
ward, the seedlings were reared in a growth chamber at ing 3/4~capacity pre-made soil and fertilizer mix (Nihon
22~ under 14 h of light at 100 nmol m -2 s-~ from white Hiryo, Japan). They were then reared in a phyt0tron under
fluorescent lamps, and at 50 to 60% RH~ Sixteen-day-old natural light conditions at 25~ with daily watering. The
seedlings were removed with forceps from the glass wool third leaves were harvested from ca. 25-day-old plants and
blocks, then weighed and immediately frozen in liquid

l:igure 1. Two (+1)-step protein extraction protocol. Schematic description of steps involved in protein extraction, starting from grinding of
leaves to very fine powder in liquid nitrogen, followed by precipitation of proteins in TCA/acetone extraction buffer (TCAAEB),treatment with
wash buffer (WB), and solubilization of precipitated protein pellet in lysis buffer supplemented with thiourea and Tris (LB-TT), then protein
determination before final 2-DGE.
 Plant Protein Extraction Protocol 415

stored at-80~ separation or stored at -80~ Equilibration of the tube gels

was clone twice with gentle agitation (15 min each) in SDS-
Extraction of Total Protein sample buffer [62 mM Tris (pH 6.8) containing 10% (v/v)
Extraction of total protein was performed through a two glycerol, 2.5% (w/v) SDS, and 5% (v/v) 2-ME]. Molecular
(+1)-step protein extraction protocol (Fig. 1). Leaf samples masses were determined by running standard protein mark-
pooled from individual seedlings were placed in liquid nitro- ers (DualColor PrecisionPhs Protein TM Standard; Bio-Rad,
gen, and ground thoroughly to a fine powder with a mortar USA).
and pestle (pre-cooled). The tissue powder (ca. 100 mg) was
transferred to sterile tubes containing TCAAEB [acetone with Pre-Cas/~PG and DALTtwelve
10% (w/v) TCA and 0.07% 2-mercaptoethanol (2-ME)]. Pro- IEF on pre-cast IPG strip gels was conducted on an IPG-
teins were precipitated for 1 h at -20~ followed by centrif- phor unit as prescribed by the manufacturer (GE Health-
ugation at 15000 rpm for 15 min at 4~ The supernatant care Bio-Sciences AB, Sweden), with some modifications to
was decanted, and the pellet was cleansed twice with wash the rehydration and IEF protocols. The volume carrying 150
buffer [acetone containing 0.07% 2-ME, 2 mM EDTA, and l~g total soluble protein was mixed with LB-TT containing
EDTA-free protease inhibitor cocktail tablets (Roche) in a 0.5% (v/v) pH 4 to 7 IPC buffer to bring it to a final volume
final volume of 100 mL buffer]. Afterward, all the acetone of 450 t~L. A trace of bromophenol blue (BPB) was added
was removed by air-drying the pellet at ambient room tem- and the entire mixture was kept at RT for 5 min, then vor-
perature (RT). The pellet was then kept at -80~ for at least texed and centrifuged at 15000 rpm for 15 min at 10~ fol-
24 h before being solubilized in lysis buffer (LB)-TT [7 M lowed by pipetting into a 24-cm strip holder tray. IPC strips
urea, 2 M thiourea, 4% (w/v) 3-[(3-cholamidopropyl) dime- (pH 4 to 7; 24 cm) were carefully placed onto the protein
thylammonio]-l-propanesulfonate, 18 mM Tris-HCI (pH samples, covered with a lid, and put into the IPGphor unit.
8.0), 14 mM trizma base, two EDTA-free protease inhibitor These strips were positioned gel-face down on the protein
cocktail tablets, 0.2% (v/v) Triton X-100 (R), and 50 mM samples to avoid air bubbles and allow for passive rehydra-
dithiothreitol (DTT), to a final volume of 100 mL]. This mix- tion with the protein samples for 1.5 h. This was followed
ture was incubated for 20 min at 4~ with occasional vor- by overlaying the IPG strips with 1.5 mL of cover fluid, a
texing and sonication, then centrifuged at 5,000 rpm for 15 process directly linked to a five-step active rehydration and
min at 10~ If further purification/clean-up of the solubi- focusing protocol (Step 1: active rehydration, step-n-hold,
lized protein samples was necessary, the supernatant from 50 V for 12 h; Step 2: step-n-hold, 100 V for 1 h; Step 3:
the last step was precipitated in 4 volumes of cold (-20~ gradient, 500 V for 4 h; Step 4: gradient, 8000 V for 12 h;
acetone and solubilized in LB-TT as above. For the seeds and Step 5: step-n-hold, 8000 V for 3 h). A total of 77000
(ca. 150 mg), LB-TT was directly added to broken seed V-h was used for the 24-cm strip, and the entire procedure
pieces and ground rapidly and thoroughly in a cold mortar was carried out at 20~ Following IEF, the IPG strips were
with pestle, followed by incubation and centrifugation of the removed from the strip holder and blotted on a Kimwipe to
homogenate as above. The supernatant was used for protein remove the cover fluid. They were then immediately used
determination with a Coomassie PlusTM (PIERCE, USA) pro- for the second dimension or stored at -20~ The strip gels
tein assay kit, and was stored in aiiquots at -80~ until ana- were incubated in 20 mL equilibration buffer [50 mM Tris-
lyzed by 2-DGE. When the sample was prepared for hand- HCI (pH 8.8), 6 M urea, 30% (v/v) glycerol, and 2% (w/v)
cast tube gels in the first dimension, 1% (v/v) ampholyte (pH SDS] containing 2% (w/v) DTT for 20 min with gentle agita-
3 to 10) was added in the LB-TT. tion, followed by incubation in the same (newly prepared
20 mL) equilibration buffer supplemented with 2.5% (w/v)
Hand-Cast Two-Dimensional Gel E[ectrophoresis iodoacetamide at RT for the same time period as above.
2-DGE was performed in hand-cast IEF tube gels on a Preceding the second dimension separation, the IPG strips
Nihon Eido vertical IEF e[ectrophoresis unit (Nihon Eido, were rinsed with cathode running buffer [0.025 M Tris,
Japan). This was followed by the second dimension using 0.192 M glycine, and 0.2% (w/v) SDS], placed on polyacry-
hand-cast polyacrylamide gels (15%) on a Nihon Eido SDS- lamide gels (DALT Cel, 12.5% of 255 x 196 x 1 mm size),
PAGE vertical electrophoresis unit (Agrawal and Rakwal, and overlaid with an overlay agarose solution [60 mM Tris-
2006). Briefly, IEF gels were made in glass tubes (3 x HCI (pH 6.8), 60 mM SDS, 0.5% (w/v) agarose, and 0.01%
5 x 14/16 (H) mm) sealed at the bottom with parafi[m. IEF (w/v) BPB]. The lower anode buffer contained 0.05 M dieth-
gel solution [28.38% (w/v) acrylamide and 1.62% (w/v) anolamine and 0.05 M acetic acid. SDS-PAGE as the second
bisacrylamide] was prepared, and loaded into the gel tubes. dimension was performed at a constant current of 2 W
After polymerization, total soluble protein (ca. 350 ~g) in overnight (ca. 14 h) on an Ettan DALTtwelve System (GE
LB-TT (maximum volume of 200 ~tL) was loaded onto the Healthcare Bio-Sciences AB) as per the manufacturer's
surface of the gel and overlaid with 20 I~L of 1/3 LB-TT. instructions. Molecular masses were determined by running
Basic (1% v/v N,N,N,N'-tetramethylethylenediamine) and standard protein markers (Bio-Rad).
acidic (0.02 N H3PO4) reservoir buffers were added, and IEF
was carried out according to the following voltage-time pro- Protein Visualization and image Analysis
gram: 200 Vfor 30 min, 400 Vfor 16 h, and 600 Vfor 1 h; To visualize the protein spots, the gels were stained with
constant voltage was used. The tube gels were either used either CBB R-250 (Fluka Chemie GmbH, Switzerland) or sil-
immediately after equilibration for the second-dimension ver nitrate. With the CBB, they were first stained for ca. 30
416 Cho et al. J. Plant Biol. Vol. 49, No. 6, 2006

rain before the excess dye was washed from the gels with a posed two modifications that improve on this protein
destaining solution to clear the background. This protocol extraction and solubilization using the original O'Farrell lysis
had been recently reviewed (Agrawal and Rakwal, 2006). [buffer (LB; O'Farrell, 1975). Their long experience in rice
For the silver nitrate, staining was carried out exactly accord- proteomics has led them to determine that an LB supple-
ing to the manufacturer's instructions for the PlusOne Silver mented with thiourea plus tris (LB-TT) dramatically increases
Staining Kit (GE Healthcare Bio-Sciences AB). Protein pat- the solubilization of proteins from the leaves of mature (two-
terns in the gels were recorded as digitalized images using a month-old, just before heading) rice (cv. Nipponbare)
digital scanner (resolution 300 dpi, greyscale, CanoScan plants. This is evidenced by a rise in the abundance of large
8000F; Canon, Japan), and saved as TIFF files. Protein spots subunit (LSU) and other medium- and high-molecular-
on the gel were quantitated in profile mode according to weight proteins, as well as the prevention of fragmentation
the operating manual for the ImageMaster 2D Platinum soft- of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)
ware (GI- Healthcare Bio-Sciences AB). compared with the performance of either LB alone or LB
supplemented with thiourea (LB-T). Their results have
revealed that even a slight modification in LB buffer compo-
RESULTS AND DISCUSSION sition can bring stability by reducing protein degradation,
along with simultaneously increasing the number of spots on
The selection of a suitable extraction buffer is the key to 2-D gels. This development has now prompted us to apply
good sample preparation; depending on the plant materials the LB-TT buffer to other model systems, such as Arabidop-
and their origin, that buffer may change for each evaluation. sis, maize, wheat (Williams et al., 2003; Puthoff et al., 2005;
Moreover, a good buffer should help one achieve two Sardesai et al., 2005a, b; Subramanyam et al., 2006), and
important goals: the extraction of all proteins in a quantita- cucumber (Kuc, 1982; Narusaka et al., 2001; Cools and
tive manner, and the protection of proteins from proteolytic Ishii, 2002). In these model plants, comparative proteomics
degradation. Agrawat and Rakwal (2006) have now pro- will be utilized to investigate plant responses to abiotic and

Figure 2. Representative2-D gel imagesof rice leaf and seed proteins separated by hand-cast IEF tube gels in first dimension. Imagesat left are
2-D gel protein profiles from young rice (cv. Nakdong) leaves extracted by directly grinding with LB-TT (A), or according to extraction protocol
presented in Figure I (B). Imagesto right are of 2.-13gel protein profiles from mature rice (cv. Nipponbare) leaves (C) and dry mature seed (D).
For each sample, ca. 350 llg of total soluble protein was loaded at cathodic end of 11 -cm-long tube gel. IEF (pH 3-I 0) and SDS-PAGE(I 5%; 12
x 14 cm)were carried out in second dimension. Proteinswere visualized by staining with CBB, and spot numbers in each gel are indicated at
bottom left-hand corners. LSU and SSU are RuBisCO large and small subunits. Black arrows mark representative protein spots increased in B
(TCAAEB/LB-TT)over A (LB-TT).
 Plant Protein Extraction Protocol 417

biotic stresses. loaded for each extraction procedure in order to compare

Using TCAAEB and LB-TT in our two (+1) protein extrac- the results by 2-DGE (Fig. 2-4). In young rice leaves, pro-
tion and solubilization protocol schematically presented teins were resolved over a pH range of 3.5 to 10.0; using
here (Fig. 1), we have demonstrated its (protocol) suitability TCAAEB prior to solubilization with LB-TT, ca. 580 CBB-
in at least five plant species (rice, Arabidopsis, maize, wheat, stained spots were detected by computerized image analysis
and cucumber) and three tissue types - leaf, seed, and software versus the ca. 482 spots seen with LB-TT alone.
crown (Fig. 2-4). This method is relatively simple, requires Mature rice leaf proteins could be nicely separated, reveal-
only a small amount of tissue (50 to 150 mg), and involves ing ca. 642 CBB-stained spots; this method provided a sig-
quickly grinding the sample to a very fine powder in liquid nificant improvement in gel profiles (Fig. 2, 5) over the
nitrogen, followed by precipitation in TCAAEB in 2 mL previous procedure of Islam et al. (2004), which also used
Eppendorf tubes. It should be noted that improper or weak TCA but recommended longer vortexing times (1 h) in the
grinding can cause problems with complete extraction of presence of glass beads. For seed samples, although
proteins and subsequent solubilization in the LB of interest. TCAAEB precipitation resulted in an insoluble precipitate
The next critical step is the washing of the resultant precipi- after the proteins were extracted from the powder, we
tates; a thorough and complete cleansing will remove any found that grinding directly in LB-TT resulted in Well-sepa-
impurities (including interfering pigments bound to the pro- rated protein patterns on 2-D gels, as evidenced by the 428
tein), and help in subsequent protein solubilization. Solubili- protein spots detected by CBB staining (Fig. 2). However, it
zation in LB-TT with occasional vortexing at 4~ for ca. 20 was also possible to precipitate the proteins extracted in LB-
min, followed by centrifugation, produces the total soluble TT by acetone (refer to additional step III; Fig. 1), thereby
protein extract. An additional precipitation step using the obtaining a more concentrated protein amount for 2-DGE
solubilized protein and cold acetone can help in further (data not shown). With Arabidopsis, we could observe even
clean-up of the protein sample, which provides good resolu- more clearly the effect of TCAAEB, in that significantly
tion in the first dimension lEE increased protein amounts were extracted from leaf samples
In testing this protocol, equal amounts of protein were after sohbilization in LB-TT (589 spots), versus LB-TT (441

Figure 3. Representative2-D gel images of Arabidopsis and maize leaf proteins separated by hand-cast IEF tube gels in first dimension (pH 3-
10). Respective images at left and right are 2-D gel protein profilesfrom young Arabidopsis (cv. Col-0) leaves and young maize (Panama c~:
Guarare 8128) leaves extracted by directly grinding with LB-TT (A, C) or by following extraction protocol presented in Figure 1 (13,D). Protein
measurements, 2-DGE method, visualization, and image analysiswere done as described in Figure 2. Black arrows mark representative protein
spots increasedin B and D (TCAAEB/LB-TT)over A and C (LB-TT).
418 Cho et al. ]. Plant Biol. Vol. 49, No. 6, 2006

Figure 4. Representative2-D gel imagesof wheat leaf, crown, and embryo, and cucumber young-leaf proteins separated by hand-cast IEFtube
gels in first dimension (pH 3-10). Firstthree 2-D gel protein profile images are of wheat young leaf (A), crown (B), and embryo (C), while image
(D) is of cucumber proteins following extraction protocol presented in Figure 1.2-DGE was carried out as in Figure2.

spots) alone (Fig. 3). A similar result was obtained with resulted from a very low amount of protein loading (150 pg)
maize leaves, where 548 spots were detected by using the compared with fewer protein spots when larger amounts of
TCAAEB plus LB-TT combination, compared with only 341 protein (350 p.g) were loaded. Moreover, the individual
spots seen from LB-TT alone (Fig. 3). Moreover, as shown by spots were highly defined and showed almost no streaking,
the individual gel images in Figure 2 and 3, the presence of indicating good resolution in the first dimension, which
large new protein spots could be easily distinguished with reflected the qualitative aspect of the LB-TT for protein solu-
the TCAAEB plus LB-TT protocol. In Figure 4, we further bilization prior to IPG. Therefore, we recommend IPG not
demonstrated that this protocol worked extremely well for only for the better resolution of protein spots (and increased
extracting and separating proteins from the leaves (684 amounts), but also for greater reproducibility and generation
spots), crowns (815 spots), and embryos (331 spots) of of 2-DGE reference maps (Zhan and Desiderio, 2003).
wheat, as well as from cucumber leaves (801 spots). Moreover, a reproducible system (large-format 2-DGE) can
Although the hand-cast IEF tube gels could separate total be applied from lab to lab in developing 2-D maps for com-
soluble protein (Fig. 2-4), it became clear that the one spot- parative proteomics.
one protein idea was redundant; significant overlaps Our study results demonstrate a simple and efficient pro-
occurred in protein spots when using the hand-cast small teomics methodology for protein extraction and 2-DGE to
size IEF tube gels (11 cm) and a non-linear pH gradient. establish reference maps of monocot and dicot sample tissues
Therefore, as Agrawal and Ral~a,al (2006) have discussed, it for applications in comparative proteomics. Using TCAEEB/LB-
was imperative to shift toward using the pre-cast linear IPG TT, we have achieved not only significant improvement in the
strips and large-format gels for 2-DGE. Here, we demon- solubilization of proteins from rice tissues, but also a dra-
strated a significant increase in the number of proteins sepa- matic alteration in protein spot numbers, quality and quan-
rated and detected in rice leaves (910 protein spots) and tity from Arabidopsis, maize, wheat, and cucumber samples
wheat crowns (907 protein spots) using large-format, pre- processed by 2-DGE. We are currently working toward the
cast 24 cm (pH 4-7) iPG strips and 26 x 20 x 1 mm poly- proteomics of those five species, with the goal of identifying
acrylamide gels, for the first and second dimensions, respec- differentially expressed proteins under the variety of envi-
tively (Fig. 5). It should be noted that these greater numbers ronmental stress conditions that plague commercial crop
 Plant Protein Extraction Protocol 419

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