protocol

Universal sample preparation method integrating

trichloroacetic acid/acetone precipitation with
phenol extraction for crop proteomic analysis
Xiaolin Wu1, Erhui Xiong1, Wei Wang1, Monica Scali2 & Mauro Cresti2
1Synergetic Innovation Center of

Henan Grain Crops, Key Laboratory of Physiological Ecology and Genetic Improvement of Food Crops in Henan Province, College of
Life Sciences, Henan Agricultural University, Zhengzhou, China. 2Department of Life Sciences, University of Siena, Siena, Italy. Correspondence should be addressed to
W.W. (wangwei@henau.edu.cn).

2014 Nature America, Inc. All rights reserved.

Published online 16 January 2014; doi:10.1038/nprot.2014.022

Crop plants contain large amounts of secondary compounds that interfere with protein extraction and gel-based proteomic analysis.
Thus, a protein extraction protocol that can be easily applied to various crop materials with minimal optimization is essential.
Here we describe a universal protocol for total protein extraction involving trichloroacetic acid (TCA)/acetone precipitation
followed by SDS and phenol extraction. Through SDS extraction, the proteins precipitated by the TCA/acetone treatment can be fully
resolubilized and then further purified by phenol extraction. This protocol combines TCA/acetone precipitation, which aggressively
removes nonprotein compounds, and phenol extraction, which selectively dissolves proteins, resulting in effective purification
of proteins from crop tissues. This protocol can also produce high-quality protein preparations from various recalcitrant tissues,
and therefore it has a wide range of applications in crop proteomic analysis. Designed to run on a small scale, this protocol can be
completed within 5 h.

INTRODUCTION
Abiotic stresses caused by global climate change, together with
biotic stresses, negatively affect crop growth and productivity
throughout the world, resulting in a reduction of crop yields
by more than 50% (ref. 1). To understand the protein networks
present in crops that respond to external stresses and increase
the tolerance of crops, comprehensive analyses of the crop proteome are required2. Comparative proteomics is a useful tool
for analyzing protein abundance between either untreated and
stress-treated samples or tolerant and intolerant crops3. Current
proteomic methods can be divided into two groups: gel-based
approaches, including conventional 2D gel electrophoresis
(2-DE) and differential in-gel electrophoresis, and gel-free mass
spectrometrybased approaches. Despite rapid advances in gelfree proteomics, 2-DE coupled with mass spectrometry is still the
primary platform for proteomic analysis4. Therefore, this protocol
focuses on the preparation of protein samples for 2-DEbased
crop proteomic analysis.
The proteomic analysis of crop plants is more problematic than
similar analyses in the model plant Arabidopsis and other organisms, because crop tissues contain large amounts of secondary
compounds such as phenolic compounds, lipids, organic acids,
carbohydrates, terpenes and pigments5,6. These compounds can
cause protein precipitation when the tissues are disrupted, and
they can severely interfere with proteomic analysis when they
co-precipitate with proteins7,8. Therefore, the extraction of highquality proteins from crop tissues is crucial for successful proteomic analyses, and a protein extraction protocol that can be
universally applied to various crop materials is essential.
Development of the protocol
Over the past two decades, tremendous efforts have been made
to develop protein extraction protocols that could enhance crop
proteomic analysis. Several quality reviews of the practical issues
associated with sample preparation have been published6,9,10.
Currently, protein extraction protocols for crop proteomic
362 | VOL.9 NO.2 | 2014 | nature protocols

analysis are typically based on TCA/acetone precipitation, phenol

extraction or a combination of these two methods.
TCA/acetone precipitation is relatively simple: plant tissues are
ground to a powder in liquid N2, the proteins are then precipitated by using either acetone or a 520% (wt/vol) TCA/acetone
solution11 and the proteins are solubilized in 2-DE rehydration
buffer. TCA/acetone facilitates the dissolution of large amounts
of secondary compounds and simultaneously precipitates the
proteins. This protocol has been successfully used with various
types of plant tissues12,13, including woody tissues8,14 and maize
leaves15,16. However, it does not produce optimal results on more
recalcitrant samples such as ripening fruits17,18 and adult olive
leaves19 compared with phenol-based methods. In addition, an
inherent drawback of this protocol is the fact that proteins may
not fully resolubilize after TCA/acetone precipitation.
Phenol extraction of proteins can be traced back to the 1950s,
when phenol was observed to be effective in extracting proteins
from aqueous solutions20. In this procedure, proteins are first
extracted by using an aqueous buffer, and the resulting extract is
then mixed with the buffered phenol (pH 7.88.0). This causes the
proteins to denature and dissolve into the phenol phase, whereas
other water-soluble substances such as salts, nucleic acids and
carbohydrates remain in the aqueous phase. Coextracted phenolic substances are then separated from the proteins after methanol precipitation. This protocol has been successfully used on
various types of plant tissues9,13,21,22, especially fruit tissues9,18.
A comparative study9 showed that both TCA/acetone- and
phenol-based methods were effective in extracting and purifying a large number of proteins, but the latter was slightly better
at removing interfering compounds from recalcitrant tissues.
However, a limitation of many phenol-based protocols is the lack
of detergents (e.g., SDS) in the extraction buffer21. Consequently,
the amount of protein extracted from the sample is often not
large enough for global proteomic analysis. In addition, phenol
extraction alone is not powerful enough to remove interfering

 2014 Nature America, Inc. All rights reserved.

protocol
compounds from more complex crop tissues such as olive leaves19,
cotton fibers23 and roots24.
To facilitate crop proteomic analyses, our group has established
sample preparation methods that are suitable for 2-DE19,2530
and studied proteomic changes in maize in response to drought
and heat stress29,3133. We designed a novel protein extraction
protocol that, for the first time, integrates TCA/acetone precipitation with phenol extraction19,26. This protocol was originally
developed to extract proteins from the adult evergreen leaves
of the olive tree (Olea europea L.), which is an economically
important oil-producing crop in many Mediterranean countries. Olive leaves contain large amounts of phenolic compounds
(~1570 mg g1 fresh weight) primarily in the form of oleuropein34. When olive leaves are destroyed by tissue disruption,
oleuropein acts as a very strong protein denaturant because it
activates enzymes (e.g., -glucosidase) in different organelles35.
This process may explain why the direct extraction of proteins
from olive leaves in aqueous extraction buffers followed by protein precipitation results in brownish pellets that are difficult
to use in downstream processes. In the case of olive leaves, this
integrated protocol is more efficient than either TCA/acetone
extraction or phenol extraction alone19.
Applications of the protocol
This protocol has been shown to have universal applications in
the production of high-quality proteins from a wide range of
crop materials. The protocol described here focuses on protein
extraction for gel-based proteomic analysis, although the proteins prepared by using this protocol can also be used in gel-free
approaches. We have shown that this protocol is suitable for a
wide range of materials, including leaves26,31,32,36, woody stems36,
roots33, fruits26, seeds28,37 and pollen38,39, which are derived from
different plant species. In addition, we have extended its application to soil metaproteome analysis40, protein desalting27 and
protein sample fractionation before 2-DE28,30 when modifications
are applied. Other researchers have also shown that this protocol
is effective for various recalcitrant tissues derived from different

plant species4162 (Table 1), although modifications must be made

depending on the types of tissue harvested and the targeted proteins to be extracted.
Comparison with other methods
There are numerous protein extraction methods based on either
TCA/acetone precipitation or phenol extraction12,13,21,22,56, many
of which are widely used in the plant (crop) proteomics field. The
distinct feature of the present protocol is that it maintains the
advantages of both TCA/acetone precipitation, which aggressively
removes nonprotein compounds, and phenol extraction, which
selectively dissolves proteins, resulting in effective purification
of proteins from crop tissues. The advantages of this protocol
are especially prominent in recalcitrant tissues. Many researchers
have compared this protocol with other methods. Jorrn et al.63
commented that it worked very well with root and leaf tissues
from Arabidopsis, Medicago truncatula and sunflowers, resulting in improved 2-DE gels compared with previous protocols.
Maldonado et al.64 showed that this protocol provided better
results with regard to spot focusing, resolved spots, spot intensity,
unique spots detected and reproducibility when compared with
the TCA/acetone precipitation and phenol extraction methods.
Similarly, Vincent et al.18 reported that this protocol resulted in
improved 2-DE gels for mature grape berry clusters.
An important aspect of this protocol is the use of SDS extraction to link the TCA/acetone precipitation with the phenol
extraction. In a classic TCA/acetone protocol, proteins are
extracted in 2-DE rehydration buffer and then directly subjected to isoelectric focusing (IEF); however, in this protocol,
proteins are extracted by using an SDS-containing buffer. With
SDS extraction, the possibility that proteins might not fully
resolubilize after TCA/acetone precipitation can be overcome,
and the resulting protein extracts can be further purified by
phenol extraction, which has also been modified for use with
various crop materials and situations. Therefore, this protocol
is different from the previously reported TCA/acetone precipitation12,13,21 and phenol extraction protocols13,22,56 (Table 2).

Table 1 | Successful applications of this protocol in various types of crop tissues.

Material

Species and references

Leaf

Maize (Zea mays)28,31,32, Chinese pistache tree (Pistacia chinensis)36, olive (Olea europaea)19,26,41, alfalfa (Medicago
sativa)42, blue carpet (Craterostigma plantagineum)43, poplar (Populus cathayana)44, cacao (Theobroma cacao)45, grape (Vitis
vinifera)46, date palm (Phoenix dactylifera)47, satsuma mandarin (Citrus reticulata blanco)48, pummelo (Citrus grandis)48,
cottonwood (Populus deltoides)49, mung bean (Vigna radiate)50, wild grapevine (Vitis pseudoreticulata)51, tobacco
(Nicotiana benthamiana)52

Stem

Chinese pistache tree (P. chinensis)36, St. Johns wort (Hypericum perforatum)53

Root

Blue carpet (C. plantagineum)43, maize (Z. mays)33, soybean (Glycine max)54, St. Johns wort (H. perforatum)53

Fruit

Upland cotton (Gossypium hirsutum) ovules55, apple (Malus domestica)26,56, banana (Musa acuminata)26, kiwi fruit (Actinidia
deliciosa)26, olive (O. europaea)26, orange (Citrus sinensis)26, pear (Pyrus calleryana)26, strawberry (Fragaria ananassa)56,
grape (V. vinifera)18,26,57, tomato (Solanum lycopersicum)26,58, aka lumbah (Curculigo latifolia)59

Seed

Legume (Lotus japonicus)60, date palm (P. dactylifera)61, maize (Z. mays)28,37

Flower

Apple (M. domestica)62, grape (V. vinifera)51, St Johns wort (H. perforatum)53

Pollen

Maize (Z. mays)38,39

nature protocols | VOL.9 NO.2 | 2014 | 363

 2014 Nature America, Inc. All rights reserved.

protocol
compounds from more complex crop tissues such as olive leaves19,
cotton fibers23 and roots24.
To facilitate crop proteomic analyses, our group has established
sample preparation methods that are suitable for 2-DE19,2530
and studied proteomic changes in maize in response to drought
and heat stress29,3133. We designed a novel protein extraction
protocol that, for the first time, integrates TCA/acetone precipitation with phenol extraction19,26. This protocol was originally
developed to extract proteins from the adult evergreen leaves
of the olive tree (Olea europea L.), which is an economically
important oil-producing crop in many Mediterranean countries. Olive leaves contain large amounts of phenolic compounds
(~1570 mg g1 fresh weight) primarily in the form of oleuropein34. When olive leaves are destroyed by tissue disruption,
oleuropein acts as a very strong protein denaturant because it
activates enzymes (e.g., -glucosidase) in different organelles35.
This process may explain why the direct extraction of proteins
from olive leaves in aqueous extraction buffers followed by protein precipitation results in brownish pellets that are difficult
to use in downstream processes. In the case of olive leaves, this
integrated protocol is more efficient than either TCA/acetone
extraction or phenol extraction alone19.
Applications of the protocol
This protocol has been shown to have universal applications in
the production of high-quality proteins from a wide range of
crop materials. The protocol described here focuses on protein
extraction for gel-based proteomic analysis, although the proteins prepared by using this protocol can also be used in gel-free
approaches. We have shown that this protocol is suitable for a
wide range of materials, including leaves26,31,32,36, woody stems36,
roots33, fruits26, seeds28,37 and pollen38,39, which are derived from
different plant species. In addition, we have extended its application to soil metaproteome analysis40, protein desalting27 and
protein sample fractionation before 2-DE28,30 when modifications
are applied. Other researchers have also shown that this protocol
is effective for various recalcitrant tissues derived from different

plant species4162 (Table 1), although modifications must be made

depending on the types of tissue harvested and the targeted proteins to be extracted.
Comparison with other methods
There are numerous protein extraction methods based on either
TCA/acetone precipitation or phenol extraction12,13,21,22,56, many
of which are widely used in the plant (crop) proteomics field. The
distinct feature of the present protocol is that it maintains the
advantages of both TCA/acetone precipitation, which aggressively
removes nonprotein compounds, and phenol extraction, which
selectively dissolves proteins, resulting in effective purification
of proteins from crop tissues. The advantages of this protocol
are especially prominent in recalcitrant tissues. Many researchers
have compared this protocol with other methods. Jorrn et al.63
commented that it worked very well with root and leaf tissues
from Arabidopsis, Medicago truncatula and sunflowers, resulting in improved 2-DE gels compared with previous protocols.
Maldonado et al.64 showed that this protocol provided better
results with regard to spot focusing, resolved spots, spot intensity,
unique spots detected and reproducibility when compared with
the TCA/acetone precipitation and phenol extraction methods.
Similarly, Vincent et al.18 reported that this protocol resulted in
improved 2-DE gels for mature grape berry clusters.
An important aspect of this protocol is the use of SDS extraction to link the TCA/acetone precipitation with the phenol
extraction. In a classic TCA/acetone protocol, proteins are
extracted in 2-DE rehydration buffer and then directly subjected to isoelectric focusing (IEF); however, in this protocol,
proteins are extracted by using an SDS-containing buffer. With
SDS extraction, the possibility that proteins might not fully
resolubilize after TCA/acetone precipitation can be overcome,
and the resulting protein extracts can be further purified by
phenol extraction, which has also been modified for use with
various crop materials and situations. Therefore, this protocol
is different from the previously reported TCA/acetone precipitation12,13,21 and phenol extraction protocols13,22,56 (Table 2).

Table 1 | Successful applications of this protocol in various types of crop tissues.

Material

Species and references

Leaf

Maize (Zea mays)28,31,32, Chinese pistache tree (Pistacia chinensis)36, olive (Olea europaea)19,26,41, alfalfa (Medicago
sativa)42, blue carpet (Craterostigma plantagineum)43, poplar (Populus cathayana)44, cacao (Theobroma cacao)45, grape (Vitis
vinifera)46, date palm (Phoenix dactylifera)47, satsuma mandarin (Citrus reticulata blanco)48, pummelo (Citrus grandis)48,
cottonwood (Populus deltoides)49, mung bean (Vigna radiate)50, wild grapevine (Vitis pseudoreticulata)51, tobacco
(Nicotiana benthamiana)52

Stem

Chinese pistache tree (P. chinensis)36, St. Johns wort (Hypericum perforatum)53

Root

Blue carpet (C. plantagineum)43, maize (Z. mays)33, soybean (Glycine max)54, St. Johns wort (H. perforatum)53

Fruit

Upland cotton (Gossypium hirsutum) ovules55, apple (Malus domestica)26,56, banana (Musa acuminata)26, kiwi fruit (Actinidia
deliciosa)26, olive (O. europaea)26, orange (Citrus sinensis)26, pear (Pyrus calleryana)26, strawberry (Fragaria ananassa)56,
grape (V. vinifera)18,26,57, tomato (Solanum lycopersicum)26,58, aka lumbah (Curculigo latifolia)59

Seed

Legume (Lotus japonicus)60, date palm (P. dactylifera)61, maize (Z. mays)28,37

Flower

Apple (M. domestica)62, grape (V. vinifera)51, St Johns wort (H. perforatum)53

Pollen

Maize (Z. mays)38,39

nature protocols | VOL.9 NO.2 | 2014 | 363

protocol

2014 Nature America, Inc. All rights reserved.

Table 2 | Comparison of the three protein extraction protocols for proteomic analysis of crop plants.
Rationale of
protein purification

Protein extraction
for 2-DE

Applications and
advantages

Tissue disruption; TCA/

acetone precipitation;
protein extraction

Remove secondary
compounds with
TCA/acetone wash
before protein
extraction

Typically uses 2-DE

lysis buffer; sometimes it is difficult
to dissolve protein
pellets

Suitable for various tissues; simple;

aggressively removes
interfering compounds,
especially lipids and
pigments

Proteins are difficult

to fully resolubilize

Phenol
extraction

Tissue disruption;
protein extraction;
phenol extraction;
protein recovery

Selectively dissolve
and purify proteins
from the aqueous
extract after protein
extraction

Typically uses a
detergent-free
aqueous buffer and
extracts only a fraction of the total
protein content

Suitable for recalcitrant

tissues; effectively
removes phenolic compounds, carbohydrates,
nucleic acids and salts

Time consuming

Protocol
reported here

Tissue disruption; TCA/

acetone precipitation;
SDS extraction; phenol
extraction; protein
recovery

Remove secondary
Total protein extraccompounds via
tion using a buffer
TCA/acetone;
containing SDS
protein purification
via phenol extraction

Universal application
in recalcitrant tissues;
has the advantages
of both TCA/acetone
precipitation and
phenol extraction

Relatively complicated and time

consuming

Protocol

Main processes

TCA/acetone
precipitation

Protein purification is more efficient when TCA/acetone precipitation and phenol extraction are integrated rather than implemented individually.
In addition, this protocol was designed to be performed on a
small scale. This approach is especially useful for comparative
proteomic analyses that require the analysis of many samples.
Owing to the small amount of starting material, overnight incubation during the TCA/acetone precipitation steps is unnecessary, resulting in a protocol that is less time-consuming than the
standard procedure.
Limitations of the protocol
This integrated protocol is somewhat complicated and time
consuming; it takes ~1 h longer than the traditional phenol
extraction. In addition, as this protocol contains many steps, small
amounts of protein may be lost. However, this loss of yield can be
remedied by performing several extractions in parallel.
Overview of the protocol
The protocol is designed to process small amounts of tissue in
Eppendorf tubes, and it can be completed within 5 h. It includes
the following five processes: tissue disruption, TCA/acetone precipitation, SDS extraction of total proteins, phenol extraction and
protein precipitation and solubilization (Fig. 1). Tissue samples
are first disrupted by using a mortar and pestle in liquid N2 and
then subjected to TCA/acetone precipitation. Consequently, substantial amounts of interfering compounds are removed via the
extensive TCA/acetone and acetone cleanup steps. Simultaneously,
the volume of the materials is greatly reduced, facilitating the use
of Eppendorf tubes in the subsequent steps. The powdered tissue is used for total protein extraction with an SDS-containing
buffer. The SDS extraction enhances the dissolution of proteins
after the TCA/acetone precipitation. Next, the protein extract is
collected and extracted with a phenol solution (pH 7.88.0) to
364 | VOL.9 NO.2 | 2014 | nature protocols

Limitations

separate the proteins from other water-soluble compounds. The

protein-containing phenol phase can be back-extracted with an
aqueous buffer to further remove interfering compounds. Finally,
the phenol phase is precipitated with 510 volumes of methanol
(containing 0.1 M ammonium acetate) to recover the proteins.
The protein pellet is then air-dried and solubilized in a buffer of
choice. The successful use of this protocol relies on the preparation of a fine tissue powder for protein extraction and the careful
recovery of the phenol phase after centrifugation. Note that some
aspects of the protocol may need to be modified, as described in
the Experimental design section. Suggestions for such modifications are included.
Experimental design
Tissue disruption. Fresh material is preferred for optimal protein extraction. Disruption of the cell wall and protein release is
crucial for the following processes. A small amount of starting
material (0.20.5 g fresh weight, two or three replicates) is recommended to ensure complete tissue disruption; small sample
sizes can provide sufficient amounts of protein for 2-DE analyses,
especially if sensitive visualization (e.g., silver or fluorescent staining) is used. One common tissue disruption technique, used in
this protocol, is to pulverize the crop materials by using a mortar
and pestle in liquid N2 (Fig. 2) or with the help of quartz sands,
which is particularly useful for fiber-rich crop tissues. The use of
fine powdered tissue as a starting material has become routine
practice in preparing samples for plant proteomics. This practice
can also minimize proteolysis and protein modifications that may
occur during tissue disruption. Alternatively, young tissues (e.g.,
root tips, watery fruit tissues and developing seeds) can be directly
homogenized in cold TCA/acetone.
TCA/acetone precipitation. This process removes most secondary compounds, particularly lipids, phenolic compounds and
pigments. A finer tissue powder results in a more thorough removal

protocol
Tissue disruption (4 C, Step 1, 20 min)
Disrupt sample in liquid N2 using a mortar and pestle

TCA/acetone precipitation (4 C, Steps 28, ~1 h)

Homogenize in TCA/acetone in the mortar
Centrifuge

Discard supernatant

Wash with TCA/acetone

Wash with acetone

Air-dry the pellet

II

SDS extraction of proteins (RT, Steps 911, ~0.5 h)

Resuspend in SDS extraction buffer
Centrifuge

2014 Nature America, Inc. All rights reserved.

Collect supernatant
Centrifuge
Collect protein extract

Phenol extraction and washes (RT, Steps 1216, ~1 h)

Mix with phenol solution (pH 8.0)
Centrifuge

II

Collect phenol phase

Wash the phenol phase
Centrifuge
Collect the phenol phase

Protein precipitation and solubilization (4 C, Steps 1724, ~1 h)

Precipitate with 0.1 M ammonium acetate in methanol
20 C, 30 min
Centrifuge
II
Wash protein pellets
II

Air-dry the protein pellets

II

Dissolve in 2-DE rehydration buffer

Protein quantification and electrophoresis separation (RT, Steps 25 and 26, ~2 d)

Bradford assay

2-DE analysis

Figure 1 | Schematic overview of the full protocol. Timing for the individual
sections of the protocol and possible pause points (shown in green) are
indicated.

of secondary compounds. Finely powdered tissue is subjected to extensive cleanup with 10% (wt/vol) TCA/acetone and acetone plus either
10 mM DTT or 2% (vol/vol) 2-mercaptoethanol (2-ME) (Fig. 2b).
Typically, after two washes each with TCA/acetone and acetone
containing 10 mM DTT or 2% (vol/vol) 2-mercaptoethanol, the
tissue pellet should ideally be white or lightly colored (Fig. 2c).
During each wash, the pellets should be fully resuspended by
either vortexing or by using a syringe with a needle. Finally, after
air-drying, the powdered tissue is ready for protein extraction.

SDS extraction of total proteins. SDS extraction is intended for

total protein extraction. This is different from the traditional
phenol extraction and TCA/acetone precipitation, which uses
detergent-free buffer to extract water-soluble proteins. In our
hands, SDS extraction produces similar protein yields when performed in duplicate, which facilitates paired comparative 2-DE
analysis. Dry powdered tissue is extracted using the SDS extraction buffer. A 0.15 M Tris-HCl solution (pH 8.8) is sufficient to
buffer any trace of TCA. The addition of bromophenol blue in
the extraction buffer is a reliable pH indicator, as it is blue under
basic conditions and yellow under acidic conditions. By using
SDS extraction, the primary issue associated with TCA/acetone
precipitation (inability of proteins to fully resolubilize) can be
overcome, and the SDS will be removed after phenol extraction. In
addition, the extract can be heated to increase protein solubilization. Protein extracts should be fully clarified by centrifugation
before phenol extraction, especially for lipid-rich materials.
Phenol extraction. Phenol and aqueous protein extracts are
immiscible. Phenol has a density of 1.07 g cm3 and therefore
forms the lower phase when it is mixed with a protein extract
(Fig. 2d). Typically, phenol and protein extract are mixed at a
1:1 (vol/vol) ratio, but this ratio can be altered depending on
the volume of the extract. After phase separation by centri
fugation, the proteins move into the phenol phase, whereas other
water-soluble substances remain in the aqueous phase. The
phenol phase is then transferred into a fresh tube and washed.
Phenol extraction can easily be performed at room temperature
(RT, 2026 C) because proteases are denatured by phenol, thus
preventing protein degradation.
After phase separation, the phenol phase usually traps ~15%
(vol/vol) of the aqueous phase. This can be back-extracted with
aqueous buffers to further remove nucleic acid and carbohydrate residues. A wash buffer containing 0.7 M sucrose forces the
phenol phase to the upper level, facilitating the transfer of the
phenol phase. Washes can also be used to reduce the volume of
the phenol phase.
Protein precipitation and solubilization. In this step, proteins
are selectively precipitated from the phenol phase with methanol, whereas phenolic substances remain soluble, thus allowing
for further protein purification. Five to ten volumes of cold
methanol plus 0.1 M ammonium acetate are added to the phenol phase, and the mixture is stored at 20 C for 30 min. A
prolonged precipitation is recommended for small amounts of
proteins. The precipitation of large amounts of proteins in a
single tube should be avoided, because the resulting pellets are
hard to resuspend and dissolve. Proteins should precipitate as
white flakes (Fig. 2e); brown or yellow pellets, which indicate the
co-precipitation of phenolic compounds, may not be suitable
for 2-DE analysis. Protein solubilization strongly affects protein
quality and quantity, and thus it determines the success of the
experiment. Protein pellets should not be overdried. In addition,
it is better to use cold 80% (vol/vol) acetone for the last wash. In
our hands, protein pellets from this protocol are always easily
dissolved in 2-DE buffer. Alternatively, protein precipitation can
be directly digested for gel-free proteomic measurements65.
Assessment of protein quality. Controls should be prepared as
described throughout the PROCEDURE to allow assessment of
protein quality and the removal of interfering compounds.
nature protocols | VOL.9 NO.2 | 2014 | 365

protocol

2014 Nature America, Inc. All rights reserved.

pH

7 4

pH

Figure 2 | Quality control and 2-DE separation

(kDa)
of maize leaf and embryo proteins. (a) Fine tissue
a
b
f
g
100
powder obtained by grinding in a mortar with
liquid N2 (Step 1). (b) Tissue powder
60
homogenized thoroughly in TCA/acetone in a
mortar (Step 2). (c) Tissue pellet (white or
c
d
e
35
lightly colored) in an Eppendorf tube after
TCA/acetone cleanup (Step 6). (d) Phase
25
separation during phenol extraction (Step 14).
15
The protein-containing phenol phase (yellowish)
forms the lower phase in an Eppendorf tube.
4
pH
74
pH
74
pH
7
(e) Protein flakes precipitated by ammonium
acetate in methanol (Step 17). (fj) Proteins
(kDa)
h
i
j
100
(~500 g) were resolved by IEF by using 11-cm
linear IPG strips (pH 47) and then separated by
using 13.5% (wt/vol) SDS/acrylamide gels. Gels
60
were stained with Coomassie brilliant blue R350.
(f) Control: direct protein extraction in 2-DE
35
rehydration buffer. Note that it did not produce
25
a sufficient protein profile with severe horizontal
streaking. (g) Control: direct protein extraction
15
with 2-DE rehydration buffer from dry powdered
tissue after TCA/acetone cleanup. Compared with
panel f, the 2-DE profile was improved, but it still showed considerable horizontal streaking. (h) Control: protein extraction with SDS-free extraction buffer
from dry powdered tissue after TCA/acetone cleanup. (i) Control: protein sample prepared without back-extraction of the phenol phase. (j) The final protein
sample prepared with this protocol, which produced an improved 2-DE protein profile with more spots compared with controls (fi).

MATERIALS
REAGENTS
2-Mercaptoethanol (2-ME; Sigma-Aldrich, cat. no. M6250) ! CAUTION
2-ME is toxic on inhalation, on contact with skin and harmful if swallowed.
Handle it with protective gloves under a fume hood.
Acetone (Sigma-Aldrich, cat. no. 650501) ! CAUTION Acetone is acutely
toxic and it is an unstable explosive. Avoid flames and handle it under
a fume hood. Dispose of acetone in accordance with relevant local
regulations.
Acid-washed sand (Sigma-Aldrich, cat. no. 18649)
Ammonium acetate (Sigma-Aldrich, cat. no. A7262)
Bio-Rad protein assay kit (Bio-Rad, cat. no. 500-0006)
BSA standards, prediluted set (Thermo Scientific, cat. no. 23208)
Bromophenol blue (Sigma-Aldrich, cat. no. B0126)
CHAPS (Sigma-Aldrich, cat. no. C3023)
Cetyltrimethylammonium bromide (CTAB; Sigma, cat. no. H6269)
DTT, for molecular biology, minimum 99% titration (Sigma-Aldrich,
cat. no. D9779)
EDTA, 0.5 M, pH 8.0 (Sigma-Aldrich, cat. no. E7889)
Fresh plant tissue (e.g., maize seeds and leaves)
Glycerol (Sigma, cat. no. G5516)
Glycine (Sigma, cat. no. 50046)
Ice
IPG buffer, pH 47 (GE Healthcare, Bio-Sciences, cat. no. 17-6000-86)
Methanol (Sigma-Aldrich, cat. no. 322415) ! CAUTION Methanol is acutely
toxic and is an unstable explosive. Avoid flames and handle it under a fume
hood. Dispose of methanol in accordance with relevant local regulations.
PMSF (Sigma-Aldrich, cat. no. P7626) ! CAUTION PMSF is acutely toxic.
Handle it with protective gloves.
Protease inhibitor cocktail (Sigma-Aldrich, cat. no. P8340)
Protein assay dye reagent concentrate (Bio-Rad, cat. no. 500-006), based on
the Bradford method
ReadyStrip IPG strips, pH 47, 11 cm (Bio-Rad, cat. no. 163-2015)
SDS (Sigma-Aldrich, cat. no. L4390) ! CAUTION SDS causes acute toxicity
and is flammable. It is a skin, eye and airway irritant. Avoid breathing dust.
Wear gloves and safety glasses when handling it.
Sodium chloride, 99.8% (NaCl; Sigma-Aldrich, cat. no. 31434)
Sucrose (Sigma-Aldrich, cat. no. S9378)
Thiourea (Sigma-Aldrich, cat. no. T8656)
Trichloroacetic acid (TCA; Sigma-Aldrich, cat. no. 522082) ! CAUTION TCA
is toxic and caustic. It may cause skin and eye burns. Wear gloves and safety
glasses when handling it.
366 | VOL.9 NO.2 | 2014 | nature protocols

Tris (Trizma base) (Sigma-Aldrich, cat. no. T1503)

Tris-buffered phenol solution (stored at 4 C, pH 7.88.0; Sigma-Aldrich,
cat. no. P4557; optionally containing 0.1% (wt/vol) 8-hydroxyquinoline)
! CAUTION Phenol may cause severe respiratory tract, skin and eye irritation
and burns; it may be fatal if inhaled. Wear gloves and safety glasses when
handling it and work under a fume hood. In case of contact, immediately
flush eyes or skin with a large amount of water for at least 10 min and seek
immediate medical attention.
Tris-HCl, 0.5 M, pH 6.8 (Bio-Rad, cat. no. 161-0799)
Tris-HCl, 1.5 M, pH 8.8 (Bio-Rad, cat. no. 161-0798)
Urea (Sigma-Aldrich, cat. no. U6504)
Water (Milli-Q, 18.2 Mcm at 25 C)
EQUIPMENT
A mortar and pestle (diameter 68 cm)
Criterion cell (Bio-Rad, cat. no. 164-6001), or similar
Eppendorf tubes (1.5 or 2.0 ml) ! CAUTION The tubes should be tested to
ensure integrity during centrifugation at 15,000g with organic solvents
(e.g., phenol).
Focusing tray, 11 cm (Bio-Rad, cat. no. 164-6111), or similar matching the
IEF device
IEF device (Bio-Rad, cat. no. 164-6001), or similar
Liquid N2 tank
Pipette and pipette tips
Power supply, universal (Bio-Rad, cat. no. 164-5070), or similar
Shaker VXR VIBRAX (IKA, cat. no. 2819000), or similar
Shimadzu UV-visible spectrophotometer (Shimadzu, UV-2550)
Sterile syringe (1.0 ml) with a needle
Temperature-controlled benchtop centrifuge (e.g., Sigma, 3-30KS)
Vortex mixer (e.g., Bio-Rad, BR-2000)
REAGENT SETUP
CRITICAL Prepare all solutions described in Reagent Setup with extreme care
to minimize contamination with keratin. Wear nonpowdered gloves at all times.
2-DE rehydration buffer Combine 7 M urea, 2 M thiourea, 4% (wt/vol)
CHAPS, 2% (vol/vol) IPG buffer, pH 47, 20 mM DTT and 0.001% (wt/vol)
bromophenol blue. This buffer may be stored without DTT for a month at
4 C or indefinitely at 80 C. It is convenient to make aliquots before freezing.
CRITICAL Add DTT just before use.
Acetone, 80% (vol/vol), in water Mix 400 ml of acetone with 100 ml of
deionized water. This solution can be stored for several months at 20 C.
CRITICAL Add DTT to 10 mM or 2-ME to 2% (vol/vol) just before use.

protocol

2014 Nature America, Inc. All rights reserved.

Ammonium acetate, 0.1 M, in methanol Dissolve 3.85 g of ammonium

acetate in 500 ml of methanol (for phenol wash only). It can be stored for
several months at 20 C.
PMSF, 100 mM Dissolve PMSF in ethanol or isopropanol. Aliquots can be
stored at 20 C for a year.
Protease inhibitor cocktail Aliquots of the commercial solution can be
stored at 20 C for 4 years. CRITICAL For all frozen aliquots, once they
are thawed, discard them after use.
SDS extraction buffer Mix 1% (wt/vol) SDS, 0.15 M Tris-HCl (pH 8.8), 0.1
M DTT, 1 mM EDTA, 2 mM PMSF, protease inhibitors (1:1,000) and 0.001%
(wt/vol) bromophenol blue (optional). This buffer may need to be modified
on the basis of the nature of the protein of interest, e.g., the use of high SDS
concentration for extraction of membrane proteins. It may be stored without

DTT for a month at 4 C or indefinitely at 80 C. It is convenient to make

aliquots before freezing. CRITICAL Add DTT and PMSF just before use.
SDS-free extraction buffer The composition of this buffer is the same as the
SDS extraction buffer but without SDS (to assess protein extraction only).
TCA, 10% (wt/vol), in acetone Dissolve 50.0 g of TCA in 400 ml of acetone.
Bring the volume to 500 ml by adding acetone. Keep the solution at 20 C
for several months. CRITICAL Add DTT to 10 mM or 2-ME to 2% (vol/vol)
just before use.
Wash buffer I Wash buffer I contains 10 mM Tris-HCl (pH 8.0), 1 mM EDTA and
0.7 M sucrose (for phenol wash only). Keep this solution at 4 C for a month.
Wash buffer II Wash buffer II contains 1% (wt/vol) CTAB, 10 mM Tris-HCl
(pH 8.0) and 0.7 M sucrose (for phenol wash only). Keep this solution at 4 C
for a month.

PROCEDURE
Tissue disruption TIMING 20 min
1| Grind two lots of 0.25 g of the tissue sample into a powder in liquid N2 by using a mortar and pestle (Fig. 2a). The
powdered tissue from the two replicates is then pooled together before the next step. Alternatively, young materials (e.g.,
root tips, developing seeds or watery fruits) can easily be disrupted without liquid N2; thus, in the case of these samples,
proceed directly to Step 2 (Table 3). A fraction of the powdered tissue (equivalent to 0.1 g of fresh tissue) should be saved
for use directly in Step 23, where it is extracted in 2-DE rehydration buffer for use as a control in Step 26. If you are
using other protein-containing liquid samples, proceed directly to Step 12; in this case, the protocol is reduced to a
phenol-based method.
CRITICAL STEP To minimize protein degradation, proceed rapidly through Steps 13 and handle the samples either on ice
or at 4 C. We recommend using a small amount of material (e.g., 0.20.5 g) in 23 replicates, which will facilitate the use
of small mortars (e.g., 68 cm). Large sample amounts are difficult to completely disrupt, making the subsequent wash and
suspension steps (Steps 57) difficult.
TCA/acetone precipitation TIMING ~1 h
2| Add 10.0 ml of cold TCA/acetone (20 C) to the mortar and homogenize the mixture (Fig. 2b). Cold acetone can
also be used in the case of young tissues. To assist with tissue disruption, 0.20.5 g of acid-washed sand can also be used
per 1.0 g of fresh material, if the tissues are rich in fiber.
CRITICAL STEP This step is the key to producing a fine tissue powder that facilitates the removal of secondary
compounds. Add the cold organic solvent while the powdered tissue remains frozen. It is convenient to homogenize the
powdered tissue first in 1.0 ml of the organic solvent and then add more.
3| Transfer the homogenate to six 2.0-ml Eppendorf tubes by using a large-bore pipette. Optionally, wash the mortar with
TCA/acetone (or acetone) and pool the washes with the homogenate.
Table 3 | Examples of recommended sample amounts, tissue disruption methods and expected protein yields for the protocol
reported here.
Tissue (amount)

Recommended tissue disruption

method

Expected protein yield

(g g1 fresh weight)

Major secondary compounds

Maize leaves (0.2 g)

In liquid N2 with a mortar and pestle

700

Phenolic compounds, pigments

Pistacia tree stem (0.5 g)

In liquid N2 with a mortar and pestle

600

Phenolic compounds, terpenes

Cotton roots (0.5 g)

In liquid N2 with a mortar and pestle

700

Phenolic compounds

Grape berry flesh (0.5 g)

In 10% (wt/vol) TCA/acetone with

a mortar and pestle

750

Phenolic compounds, lipids, pectin,

organic acids

Dry maize embryo (0.1 g)

In 10% (wt/vol) TCA/acetone with

a mortar and pestle

850

Carbohydrates, lipids

Maize pollen (50 mg)

In 10% (wt/vol) TCA/acetone with

a mortar and pestle

800

Phenolic compounds, lipids, pigments

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protocol
4| Centrifuge the tubes at 15,000g for 5 min at 4 C to collect the precipitated proteins. Discard the supernatant.
CRITICAL STEP There is no need to change the tubes during Steps 48. If sand is used in Step 2, it will be present in the
pellet.
CRITICAL STEP Samples are centrifuged for a relatively short time to avoid a longer TCA/acetone incubation during this
step. Separate the protein pellets from the supernatant, which contains substantial amounts of secondary compounds, as
soon as possible to minimize protein modification.
5| Resuspend the pellet in 1.01.5 ml of cold TCA/acetone (or acetone) in each tube and centrifuge the tubes as described
in Step 4.
CRITICAL STEP The tissue pellet volume should not exceed 10% of the volume of each Eppendorf tube, thus facilitating
thorough washes in the following steps.
CRITICAL STEP Be sure to thoroughly break up the pellets by pipetting, vortexing or drawing in and out of each pellet
with a 1.0-ml syringe and needle.

2014 Nature America, Inc. All rights reserved.

6| Repeat Step 5 (usually two times) until the pellet becomes white or lightly colored (Fig. 2c).
7| Resuspend each pellet in 1.5 ml of cold acetone and centrifuge it at 15,000g for 5 min at 4 C. Discard the supernatant
and repeat this acetone wash step one more time.
CRITICAL STEP Pellets should be completely suspended by pipetting or vortexing to fully remove residual TCA. The mixture
should become a uniform suspension without large flakes and particles.
8| Collect the final pellet. Allow the pellet to completely dry in a fume hood to ensure that all of the acetone has
evaporated. A fraction of the pellet (equivalent to 0.1 g of fresh tissue) should be saved for use directly in Step 23, where it
is processed for use as a control in Step 26.
CRITICAL STEP The dry pellet should ideally be white or lightly colored with no unbroken tissue pieces; otherwise, grind
the dry pellet in a mortar again and repeat Steps 28 once.
? TROUBLESHOOTING
PAUSE POINT The dry powdered tissue can be used immediately for protein extraction or it can be stored between 20 C
and 80 C for several months.
SDS extraction of total proteins TIMING ~0.5 h
9| Resuspend the dry powdered tissue from Step 8 in SDS extraction buffer (~1.5 ml per 0.1 g of fresh tissue). Extract a
fraction of the dry powdered tissue (equivalent to 0.1 g of fresh tissue) in SDS-free extraction buffer for use as a control in
Step 26. Alternatively, the dry powdered tissue can be homogenized in a mortar in the extraction buffer to enhance protein
dissolution. Transfer the homogenate to Eppendorf tubes (e.g., 1.3 ml per 1.5-ml tube; 1.8 ml per 2.0-ml tube) by using a
large-bore pipette and incubate it at RT or between 60 and 70 C for 12 h.
CRITICAL STEP The use of a mild alkaline solution (0.15 M Tris-HCl, pH 8.8) assures full neutralization of any TCA residue.
Extraction in the presence of SDS can be performed at either RT or higher temperatures.
? TROUBLESHOOTING
10| Centrifuge the tubes at 15,000g for 10 min at RT and transfer the resulting supernatant into new Eppendorf tubes
(e.g., 600 l per 1.5-ml tube; 800 l per 2.0-ml tube).
11| (Optional) The resultant pellet from Step 10 can be extracted once more by adding 0.5 ml of SDS extraction buffer per
tube and repeating the centrifugation in Step 10; then, the supernatant can be pooled with the supernatant collected in
Step 10.
CRITICAL STEP High-speed centrifugation is recommended to obtain a clarified extract.
Phenol extraction and washes TIMING ~1 h
! CAUTION Throughout the steps given below involving phenol, be sure to wear gloves, to keep tubes closed and to handle
the solutions in a fume hood.
12| Add an equal volume of Tris-buffered phenol to the protein extracts from Step 10. Note that depending on the volume
and type of the protein extract the ratio of the protein extract to phenol may vary in some cases. For larger volumes
(e.g., 510 ml) of diluted protein extract (e.g., dialysate and elute), the extract can be mixed with a 0.1- to 0.5-fold volume
of phenol in a large centrifugation tube to concentrate the proteins; for smaller volumes (<200 l), the protein extract
368 | VOL.9 NO.2 | 2014 | nature protocols

protocol
(e.g., tissue bleeding sap) can be diluted with distilled water into a larger volume (e.g., 500 l) before being mixed with
phenol (1:1, vol/vol) to minimize protein loss.
CRITICAL STEP Check the pH of the protein extract before mixing it with phenol. Take 1 l of the sample and pipette it
onto a piece of pH paper. The pH should be between 7.0 and 9.0. If the pH is <7, add drops of 0.5 M Tris base; if the pH is
too basic, add drops of 0.5 M glycine.
CRITICAL STEP Make sure to pipette the correct phenol phase from a commercial phenol (pH 7.88.0) bottle. The phenol
phase is at the bottom, and it is yellowish in color with 0.1% (wt/vol) 8-hydroxyquinoline and colorless without
8-hydroxyquinoline.
13| Mix the two parts well by shaking or vortexing for a short period time (e.g., 35 min). The mixture will become an emulsion.
PAUSE POINT This shaking step can be prolonged for 12 h at RT at a speed of 180 r.p.m.

2014 Nature America, Inc. All rights reserved.

14| Centrifuge the mixture at 15,000g for 5 min at RT. The mixture will divide into two phases with an interface (Fig. 2d).
? TROUBLESHOOTING
15| Collect the phenol phase containing proteins in new Eppendorf tubes. Process 1.0 ml of the phenol phase without
back-extraction, by proceeding to Step 17, for use as a control in Step 26.
CRITICAL STEP The phenol phase forms the lower phase. However, it will form the upper phase if the protein extract is
dense owing to high sucrose (e.g., 0.7 M) or salt (>0.5 M) concentrations. The phenol phase is easily identified by its yellow
color due to 8-hydroxyquinoline that is added to the phenol during equilibration. In the presence of bromophenol blue, the
phenol phase is bluer than the aqueous phase.
CRITICAL STEP Be careful to collect the phenol phase by using a pipette or syringe. Do not disturb the interface or the
pellets (if any). It is convenient to use a 1.0-ml syringe with a needle to collect the lower phenol phase.
16| Add an equal volume of wash buffer I to the phenol phase and mix it well, as described in Step 13. Repeat the
centrifugation, as described in Step 14. Collect the phenol phase (the upper phase). (Optional) For carbohydrate-rich tissues
(such as endosperms, ripening fruits, pollen and citrus leaves), the phenol phase is washed once more with wash buffer II.
? TROUBLESHOOTING
Protein precipitation and solubilization TIMING ~1 h
17| Pipette the phenol phase into fresh Eppendorf tubes (200 l in each tube) and add 0.1 M ammonium acetate in
methanol to a total volume of 1.5 or 2.0 ml (depending on the volume of the Eppendorf tube). Mix it thoroughly by shaking
or vortexing for 30 s, and then store it at 20 C for 30 min to precipitate the phenol-extracted proteins. Ideally, proteins
precipitate as white flakes (Fig. 2e). Brown or yellow pellets may be not suitable for 2-DE analysis.
CRITICAL STEP Use at least five volumes of 0.1 M ammonium acetate in methanol to precipitate the proteins.
Alternatively, five volumes of acetone can be used to precipitate small amounts of proteins. Avoid using sucrose in Step 16
when using acetone precipitation, as the sucrose partitions into the phenol phase and acetone will precipitate it.
PAUSE POINT Usually protein pellets become visible within several minutes. An overnight incubation is preferable for a
small amount of proteins.
18| Centrifuge the mixture at 15,000g for 10 min at 4 C. Proteins will be precipitated and phenolic substances will dissolve
in the phenol/methanol mixture. Discard the supernatant.
! CAUTION The supernatant contains toxic phenol and should be disposed of appropriately.
? TROUBLESHOOTING
19| Wash the protein pellets by adding 1.01.5 ml of 0.1 M ammonium acetate in methanol. Resuspend the pellets
thoroughly by drawing in and out of the pellet with a 1.0-ml syringe and needle. Centrifuge the mixture at 15,000g for
5 min at 4 C. Discard the supernatant.
20| Wash the protein pellets by adding 80% (vol/vol) acetone (1.01.5 ml per tube). Resuspend the pellets thoroughly by
drawing in and out of each pellet with a 1.0 ml-syringe and needle. Centrifuge the suspensions at 15,000g for 5 min at 4 C.
Discard the supernatants. This washing step should completely remove the phenol, methanol and ammonium acetate, and it
should help prevent the protein from drying excessively.
PAUSE POINT The pellets can be stored at 20 C for a month.
21| Repeat Step 20 once.
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protocol
22| Air-dry the protein pellets at RT.
CRITICAL STEP Do not overdry the protein pellets. A completely dry pellet is difficult to resuspend in 2-DE rehydration
buffer. Therefore, monitor the pellet as it dries. Approximately 35 min of air-drying is usually needed.
? TROUBLESHOOTING
PAUSE POINT The pellets can be stored at 20 C or at 80 C for a month.
23| Resuspend each final protein pellet in an appropriate volume (e.g., 100200 l) of 2-DE rehydration buffer.
Alternatively, the protein pellet can be directly digested with a protease for bottom-up proteomic analysis without having to
resolubilize the proteins. The sample can be incubated for 12 h at RT with agitation.
CRITICAL STEP Do not heat the protein sample in the presence of urea, as these conditions will lead to the carbamylation
of the proteins.
CRITICAL STEP Protein solubilization is crucial for successful 2-DE. In our hands, the protein pellets from this protocol are
easy to dissolve in 2-DE rehydration buffer. If necessary, increase the volume of buffer to fully resolubilize the proteins.
? TROUBLESHOOTING

2014 Nature America, Inc. All rights reserved.

24| Centrifuge the suspensions at 15,000g for 10 min at 1525 C. Collect the supernatant.
Protein quantification and electrophoretic separation TIMING ~2 d
25| Quantify the protein extract by the Bradford assay66.
CRITICAL STEP Protein concentration must be determined at this step for 2-DE to load a sufficient protein amount into
the IPG strip; the amount of protein loaded depends largely on the gel visualization method (e.g., Silver stain, 50100 g per
11 cm strip; Coomassie brilliant blue R or G, 400600 g per 11 cm strip). If a low protein yield <200 g g1 fresh tissue) is
obtained, we recommend using more sample replicates (e.g., three or four, depending on the nature of the crop tissues).
? TROUBLESHOOTING
26| Perform the protein separation by 2-DE to allow quality control of the extracted proteins or comparison of protein profiles among protocols, as described in the ANTICIPATED RESULTS. Dissolve proteins (~500 g) in 200 l of 2-DE rehydration
buffer and load it via overnight rehydration into 11-cm linear IPG strips (pH 47). Perform both the first-dimension IEF and
the secondary SDS-PAGE separations according to standard laboratory practices7.
CRITICAL STEP For crop tissues, the IEF time should be prolonged empirically. For example, we focused on proteins
from crop tissues in 11-cm IPG strips (Bio-Rad) with a total of 80,000100,000 volt-hours, which is much larger than the
recommended 20,00040,000 volt-hours.
? TROUBLESHOOTING
? TROUBLESHOOTING
Troubleshooting advice can be found in Table 4.

Table 4 | Troubleshooting table.

Step

Problem

Possible reason

Solution

The powder is yellowish

or brownish, not white or
lightly colored

Grinding is not sufficient, and there are

unbroken tissue pieces in the powdered
tissue
Washes are not fully sufficient

Grind the powdered tissue in a mortar again and

repeat Steps 28 once time

Fresh materials are not immediately

processed or are not frozen after sampling;
the samples were stored at 20 C instead
of 80 C, resulting in oxidation during
tissue disruption and storage
9

The protein extract is

yellow in the presence of
bromophenol blue,
indicating an acidic pH

TCA residue is present in the dry powdered

tissue

Increase wash times; fully suspend the pellet during

each wash step. If necessary, use a syringe to fully
suspend the pellet
Use fresh materials and rapidly process the sample in
liquid N2 during tissue disruption; quickly wash using
cold TCA/acetone; increase the concentration of the
reducing agent (e.g., DTT or 2-ME)
Use drops of 0.5 M Tris base solution or 1.5 M Tris-HCl
(pH 8.8) to neutralize

(Continued)
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protocol

2014 Nature America, Inc. All rights reserved.

Table 4 | Troubleshooting table (continued).

Step

Problem

Possible reason

Solution

14,16

No phase separation or
abnormal phase separation occurred after
centrifugation (e.g., little
phenol phase forms)

The aqueous phase rather than the phenol

phase was removed from the phenol bottle
during phenol separation
SDS concentration in the extraction buffer
is too high, e.g., >5% (wt/vol)
The proteins were extracted using 2-DE
rehydration buffer; urea, thiourea and
CHAPS interfere with phase separation
Organic solvents or large amounts of
phenolic compounds are in the powdered
tissue. These can interfere with phase
separation
Centrifugation was not sufficient
Temperature was too low for phenol
extraction

The phenol phase is denser than the aqueous phase;

make sure to pipette the phenol phase, which is the
lower phase in a bottle
Decrease SDS concentration in the extraction buffer to
1% (wt/vol)
Protein extraction with 2-DE rehydration buffer is
not compatible with phenol extraction. Use another
aqueous buffer
Ensure that the starting sample does not contain
organic solvents (e.g., methanol or DMSO), alkaline
reagents or soluble PVP. Extensively wash using
acetone and air-dry before protein extraction
Centrifuge at 20,000g for 10 min
Perform phase separation and centrifugation at 4 C
and above (e.g., RT)

18

Poor protein yield (little

or no protein precipitation)

The incorrect phase was removed for

protein precipitation
Methanol alone, without 0.1 M ammonium
acetate, was used
The time of the protein precipitation was
short at 20C
Centrifugation was not sufficient
Sample has a low protein content

Ensure that the correct phenol phase is removed

Extraction was not sufficient; e.g., a

detergent-free extraction buffer was used
The pH of the extraction buffer or phenol
was much lower than 7
A small amount of tissue sample was used

Ensure that the methanol contains 0.1 M ammonium

acetate
Precipitate for a longer time at 20C or if necessary,
precipitate overnight
Centrifuge at a higher speed, for longer time, or both
Increase the sample amount and precipitate proteins
with acetone
Use an SDS-containing buffer to homogenize the sample in a mortar (adding sand); heat if necessary
Adjust the pH above 7 before phenol extraction
Use more tissue sample

22

The protein pellet is

yellowish or brownish

Proteins co-precipitated with phenolic

compounds and oxidized

These proteins are not suitable for 2-DE. It is better to

re-extract the proteins

23

The proteins do not

dissolve well

The protein pellet was overdried; the

volume of 2-DE rehydration buffer was
not sufficient or contains no thiourea

Do not air-dry the protein pellets too long, and be

sure to monitor the drying process. Use more 2-DE
rehydration buffer with 2 M thiourea

25

The protein assay gives

inaccurate (abnormally
high) or inconsistent
results

The protein sample contains components

(e.g., SDS, IPG buffer, -ME or bromo
phenol blue) that may interfere with
protein quantification assays

Use a precipitation step (e.g., TCA or acetone

precipitation) to remove any interfering substances
before quantification.
Alternatively, dissolve the proteins in 2-DE rehydration
buffer without these components before the Bradford
assay. After the assay, supplement these components
as required

26

Horizontal or vertical
streaking is present on
the gel

Problems occurred during IEF or SDS-PAGE

Refer to the troubleshooting guide for the 2-DE

system in the technical bulletins 2D Electrophoresis:
Principles and Methodsa (GE Healthcare) or 2D
Electrophoresis for Proteomicsb (Bio-Rad)

Only a few protein spots

are present on the 2-DE
gels

Not enough protein was loaded

The staining was not sensitive enough

Increase the amounts of protein loaded on the IEF strips

Use more-sensitive methods for staining (e.g., silver
or fluorescent staining)

Note: Websites for free download.

ahttp://www.nubiomol.ufv.br/protocolos_files/2D%20Handbook3.pdf. bhttp://www.bio-rad.com/LifeScience/pdf/Bulletin_2651.pdf.

nature protocols | VOL.9 NO.2 | 2014 | 371

protocol
TIMING (To simultaneously prepare two samples)

2014 Nature America, Inc. All rights reserved.

Step 1, tissue disruption: 20 min

Steps 28, TCA/acetone precipitation: ~1 h
Steps 911, SDS extraction of total proteins: ~0.5 h
Steps 1216, phenol extraction and washing: ~1 h
Steps 1724, protein precipitation and solubilization: ~1 h
Steps 25 and 26, protein quantification and electrophoretic separation: ~2 d

ANTICIPATED RESULTS
This protocol adopts TCA/acetone and acetone cleanup steps to remove most of the interfering substances, and a colorless
or white tissue pellet is obtained at Step 8 (Fig. 2c). When analyzing recalcitrant materials (e.g., grape berry), the powered
tissue may be reddish. This pigmentation does not significantly affect the subsequent extraction quality. The final protein
precipitate (Step 22) should be colorless or white (Fig. 2e). A yellowish or brownish pellet is probably not suitable for 2-DE.
This type of pellet is common when a large amount of recalcitrant crop tissue is used, resulting in incomplete tissue disruption and the coextraction and co-precipitation of phenolic substances and proteins.
Maize embryos and leaves were used as examples to assess the utility and practicality of the present protocol. Quality
control was assessed via 2-DE in the pH range of 47 (Step 26). The use of narrow pH gradients increases the resolution and
facilitates comparative studies. The effects of TCA/acetone precipitation (Fig. 2f,g), protein extraction (with or without SDS,
Fig. 2h,j) and back-extraction of phenol phase (Fig. 2i,j) on protein profiles of maize embryos were compared by 2-DE. The
methods for preparing controls are described in Steps 1, 8, 9 and 15. Direct protein extraction in 2-DE rehydration buffer did
not produce a sufficient protein profile, with severe horizontal streaking (Fig. 2f). After the TCA/acetone cleanup, protein
profile was improved but still with considerable horizontal streaking (Fig. 2g). The SDS-free extraction produced fewer spots
than the SDS extraction (Fig. 2h versus Fig. 2j). The quality of the protein profile was partly improved by back-extraction
of the phenol phase (Fig. 2i versus Fig. 2j). The above results indicate that improved 2-DE maps of maize embryos (Fig. 2j)
can be obtained by combining TCA/acetone precipitation and phenol extraction.
In addition, two classical extraction protocols, TCA/acetone precipitation12 and phenol extraction9, were used as controls.
Maize leaf proteins extracted by the TCA/acetone precipitation, phenol extraction methods and the protocol described here
were compared via 2-DE (Fig. 3). All of the proteins were dissolved in the same 2-DE rehydration buffer, and they were qualitatively and quantitatively compared via 2-DE on equal protein-loading basis. Protein yields from the two control protocols
and our protocol were compared by using the Bradford assay (Step 25). For young maize leaves, the average yield was
3.6 mg g1 fresh weight for our protocol, 3.9 mg g1 fresh weight for the phenol extraction protocol and 4.5 mg g1 fresh
weight for the TCA/acetone precipitation. Among the three protocols, the TCA/acetone precipitation method yielded the
highest protein content, but its corresponding 2-DE gel had horizontal streaks, indicating the presence of nonprotein substances. Therefore, the Bradford assay does not accurately quantify the true protein content. A previous comparison indicated that there were no statistically significant differences in the protein yield among the three protocols64.
In addition, we compared this
protocol with the two control pro4
pH
7 4
pH
7 4
pH
7
tocols by using the fifth leaves and
(kDa)

100

Figure 3 | Comparative 2-DE analysis of maize

leaves proteins by using three extraction
protocols. (ac) 2-DE maps from the fifth
leaves of young maize seedlings. (df) 2-DE
maps from the 15th leaves of adult maize
plants grown in the field. Compared with the
fifth leaves, the 15th leaves are recalcitrant
to disrupt and contain more interfering
substances. (a,d) Proteins extracted with the
protocol reported here. (b,e) Controls: proteins
extracted with the phenol extraction protocol.
(c,f) Controls: proteins extracted with the
TCA/acetone precipitation protocol. 2-DE maps
(a,d) produced by using our protocol were
better than those (b,c,e,f) from the two control
protocols. Proteins (~500 g) were resolved by
IEF by using 11-cm linear IPG strips (pH 4-7)
and then separated by using 13.5% (wt/vol)
SDS/acrylamide gels. Gels were stained with
Coomassie brilliant blue R350.
372 | VOL.9 NO.2 | 2014 | nature protocols

60

35
25
15

4
(kDa)
100
60

35
25
15

pH

7 4

pH

7 4

pH

protocol
the 15th leaves (more recalcitrant) of maize plants grown in the field conditions. Similar 2-DE profiles were obtained from
young leaves by using all three protocols, and our protocol produced slightly better results (Fig. 3ac). The advantage of
our protocol was obvious on the adult leaves (Fig. 3df). The differences in the 2-DE profiles were caused by the extraction
methods themselves, as the same 2-DE rehydration buffer and equal protein loading were used for the comparison. From the
young leaf extracts, the average number of spots detected by PDQUEST software was 603 for our protocol, 562 for the phenol
extraction protocol and 595 for the TCA/acetone precipitation. These results are only a small representation of the maize
proteome. Obviously, no single method can extract the complete proteome.
Acknowledgments Work in our laboratory was supported by grants from
the National Natural Science Foundation of China (grant nos. 31230055 and
31371543) and the Ministry of Science and Technology of China (grant no.
2011CB111509).

2014 Nature America, Inc. All rights reserved.

AUTHOR CONTRIBUTIONS X.W. and E.X. conducted the experiments and

interpreted the data. X.W. and W.W. developed the protocol, interpreted the data
and drafted the manuscript. M.S. and M.C. developed the sections of the protocol
and provided intellectual and technical expertise.
COMPETING FINANCIAL INTERESTS The authors declare no competing financial
interests.
Reprints and permissions information is available online at http://www.nature.
com/reprints/index.html.
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