Extraction of Proteins from Plant Tissues

Proteins are extracted from plant tissues for a wide range of reasons, including to assay
an enzyme in a crude extract for physiological studies, to purify a protein in order to
identify the gene that encodes it, and to resolve plant proteins by SDS-PAGE. Plants
differ from animals in that they have a rigid cellulose cell wall, which may be further
strengthened by lignification. In addition, they often have a large vacuole that contains
segregated secondary plant products (especially phenolics and polyphenolics), organic
acids, and proteinases. These vacuoles are broken upon grinding, releasing contents that
may modify, inactivate, precipitate, or degrade proteins. Consequently, special techniques
are required to disrupt the cell walls and to protect proteins from damaging components
released on breakage (see Critical Parameters).

UNIT 4.7
BASIC
PROTOCOL

Materials
Frozen or fresh plant material
Liquid nitrogen
Extraction buffer (see recipe)
Mortar and pestle or tissue grinder (Freezer/mill from SPEX CertiPrep or
equivalent)
20- to 70-m pore size nylon mesh (screen printing mesh) or Calbiochem
Miracloth for filtration
Small paintbrush
Refrigerated centrifuge and centrifuge tubes
NOTE: All operations at should be carried out at 0 to 4 C (i.e., in a cold room, or on ice)
unless there are reasons to do otherwise. The extraction procedure should be performed
rapidly to minimize exposure of proteins of interest to potentially damaging compounds
and enzymes released upon cell breakage.

Disrupt tissue
1. Grind frozen plant material to a fine powder using a mortar and pestle prechilled to
186 C in liquid nitrogen, or in a tissue grinder designed to work with liquid nitrogen.

It is easiest to grind without having the liquid nitrogen come into contact with the tissue. To
ensure that the sample stays frozen, the mortar may be placed in a shallow pool of liquid
nitrogen (e.g., in the lid of a standard polystyrene box).
Fresh material may be ground directly in ice-cold buffer. A tissue homogenizer is especially
effective for grinding fresh plant material. Other options include a blender or a mortar
and pestle plus acid-washed sand. Foaming can be a problem when blending, and some
measure of control may be achieved by adding a few drops of n-octanol.
CAUTION: Wear gloves and safety glasses to avoid burns while working with liquid
nitrogen.

2. Transfer the frozen sample powder to ice-cold extraction buffer, mix quickly, and grind
further with a chilled mortar and pestle or a tissue homogenizer.
In most cases, use a tissue/buffer ratio equal to 1:5 (w/v) (see Critical Parameters). Do not
add buffer to the 186 C mortar and pestle, as the sample will then take a very long time
to thaw. Instead, add the frozen powder to the extraction buffer in an ice-cold container
and mix the buffer into the powder quickly, before thawing of the powder occurs. Use a
paintbrush to aid the transfer.
Extraction,
Stabilization,
and
Concentration
Contributed by William Laing and John Christeller
Current Protocols in Protein Science (2004) 4.7.1-4.7.7
C 2004 by John Wiley & Sons, Inc.
Copyright

4.7.1
Supplement 38

Purify extract
3. Filter the extract immediately through a 20- to 70-m nylon mesh (as used in screen
printing) or Miracloth, squeezing by hand to remove cell walls and other debris.
For particularly large volumes, a press, such as a hand-pumped cider press, can be used.

4. To remove insoluble material from the filtered sample, centrifuge large-volume extracts >10 min at 30,000 g, 4 C; microcentrifuge small-volume extracts 10 min at
maximum speed, 4 C.
5. Decant the supernatant carefully, watching out for an unstable pellet.
Chlorophyll will be solubilized if there is a detergent in the extraction buffer (see Reagents
and Solutions); if no detergent is present, most chlorophyll will be in the pellet. A white
precipitate may be present, consisting of starch and/or insoluble polyvinylpyrrolidone.

6. If quantitative recovery is important, extract the pellet again with more buffer, filter,
and centrifuge again. Combine the supernatants.
7. Depending on the purpose of the experiment, perform additional standard protein
purification steps (see Chapter 4) and/or direct analysis by SDS-PAGE (UNIT 10.1) on
the supernatant.
Alternatively, if the protein is being extracted for resolution by SDS-PAGE (Schagger and
von Jagow, 1987), the tissue can be ground until finely powdered (as described in step 1), a
small amount (e.g., 50 mg) quickly weighed and placed into a tube, and a suitable volume
of hot SDS sample buffer added. This is mixed and immediately heated to denature proteins,
centrifuged, and applied to the gel.

REAGENTS AND SOLUTIONS

Use Milli-Q-purified water or equivalent for the preparation of all buffers. For common stock
solutions, see APPENDIX 2E; for suppliers, see SUPPLIERS APPENDIX.

Extraction buffer
0.2 M 3-(N-morpholino)propanesulfonic acid (MOPS), pH 7.0
5% (w/w) polyvinyl polypyrrolidone (PVPP; weigh out, add to mixture, and allow
to hydrate 1 hr before use)
1% (v/v) Triton X-100 (optional; see Critical Parameters)
10% (v/v) glycerol (optional; see Critical Parameters)
Store buffer with above components up to several weeks at 4 C
On day of use, add the following:
2 mM dithiothreitol (DTT)
Suitable plant proteinase inhibitor cocktail (Sigma, Roche, or equivalent; add
according to manufacturers instructions, or 30 min before tissue extraction;
see Critical Parameters)
COMMENTARY
Background Information

Extraction of
Proteins from
Plant Tissues

There are two main purposes for extracting proteins from plant tissues: to assay protein levels or activity in a crude extract, or
to purify relatively large quantities of an enzyme or protein. The methods chosen thus
depend on purpose, tissue type, and enzyme
or protein to be extracted. In this unit, various options are discussed for making appropriate modifications to cope with a particular

situation. The more that is known about the

properties of the protein of interest, the easier it is to make choices regarding extraction
methods.
Generally, the better the health of the plant
tissue, the greater the activity that will be extracted (unless the target protein is a stress- or
disease-related protein). In addition, younger
plants and unstressed tissues often have fewer
interfering compounds.

4.7.2
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Current Protocols in Protein Science

Critical Parameters
Extraction buffer
Buffer is required to maintain stability
of enzymes from both a pH and an ionicstrength standpoint. Because plant cytoplasm
is around pH 7 and chloroplasts are at a
higher pH, it is generally optimal to maintain extracted enzymes at a similar, neutral
pH. However, extraction at pH > 7 is not recommended (see below). Alternative buffers to
MOPS include N-2-hydroxyethylpiperazineN -2-ethanesulfonic acid (HEPES), phosphate, or 2-(N-morpholino)-ethanesulfonic
acid (MES), depending on the goals of the
experiment and the need to avoid particular
buffers. As the contents of plant cell vacuoles
are often quite acidic, sufficient buffering capacity should be used to ensure the pH does
not drop below the acceptable range for the
protein of interest.
The volume of extraction buffer used per
unit weight of tissue depends on the tissue and
the purpose of the extraction: e.g., the need for
complete protein extraction versus the convenience of small volumes. Better extraction is
obtained with a higher ratio of buffer volume
to fresh tissue weight (e.g., 10 to 1). For largescale extractions, however, this often produces
too large a volume to work with in subsequent
steps unless the sample is first concentrated.
An insufficient volume ratio of buffer risks
changes in the pH of the extract and modification of the proteins in the tissues.
A buffer containing 10% to 20% glycerol
(v/v) may be necessary to ensure the stability of
particularly unstable proteins. Some proteins
(e.g., cell wall proteins and some seed storage
proteins) are only solubilized in the presence
of high salt concentrations, in the range of 0.1
to 1 M (Mika and Luthje, 2003).
Antioxidants
Antioxidants in the extraction buffer are essential to maintain the reduced state of free
sulfhydryl groups in enzymes and to reduce
oxidation of other components, such as phenolics. Options include DTT (2 mM or more),
-mercaptoethanol (5 to 10 mM), or ascorbic acid (5 to 10 mM), and in some circumstances, stronger reducing agents such as
sodium dithionite (Melville and Ryan, 1972).
More than one reducing agent can be used
(e.g., DTT and -mercaptoethanol). Antioxidants also reduce the activity of polyphenol oxidases that, in the presence of oxygen, rapidly
synthesize polyphenols from phenolics upon
tissue disruption. Polyphenol oxidase (PPO)

inhibitors, such as sodium diethyldithiocarbamate (1 to 5 mM) are effective against

copper-containing PPOs. As plant tissues are
often naturally high in ascorbic acid, it should
be noted that ascorbic acid can be oxidized
in the presence of metals, producing hydrogen peroxide, which can oxidize cysteine
(Schmalhausen et al., 2003). The iron chelator deferoxamine is effective at stopping this
oxidation, whereas EDTA actually promotes it
(Schmalhausen et al., 2003).
Detergents
Detergents are used to disrupt membranes
and are therefore essential for solubilizing
membrane proteins (Mika and Luthje, 2003).
However, they may also be useful for maximizing the yield of soluble proteins in some
circumstances, especially when low ratios
of extraction buffer to tissue are used. A
series of detergents or other chaotropes can
be used to carry out differential extractions.
Examples include Tween 80 (0.1% to 1%),
Triton X-100 (0.1% to 1%), or sometimes
3-[(3-cholamidopropyl)dimethylammonio]2-hydroxy-1-propanesulfo-nate (CHAPS) or
octyl glycosides. Generally, initial empirical
testing is needed to determine optimal
combinations.
Protective agents
Protective agents are required to prevent
polyphenols such as tannins (water-soluble
polyphenols of varying molecular weight that
are the most abundant polyphenols; Spencer
et al., 1988; Singh et al., 2003) from binding to and inactivating proteins. The insoluble
PVPP or soluble polyvinyl pyrrolidone (PVP)
are useful in most applications (Loomis, 1974).
These bind polyphenols best at pH values below 7 (Makkar et al., 1995). PVP is useful
when making organelles or otherwise separating samples from the medium (by centrifugation or chromatography) early in the procedure. PVPP has the advantage that it is removed from the medium at the filtration and
centrifugation steps. PVPP (0.5 to 5%, w/v)
must be hydrated for 1 hr in the extraction
buffer before use. Polyethylene glycol (e.g.,
PEG 6000) is also very useful for binding
polyphenols.
Proteinase inhibitors
Proteinase inhibitors (PIs) are essential
for controlling degradation of proteins following maceration. Cysteine proteinases are
most widespread in plant tissues but metallo-,
serine, and aspartic proteinases have been
reported in different tissues. Furthermore, a

Extraction,
Stabilization,
and
Concentration

4.7.3
Current Protocols in Protein Science

Supplement 38

buffer pH of around 7 is conducive to cysteine

proteinase activity, as is the presence of antioxidants. Some proteinase inhibitors are irreversible and chemically modify their targets
(e.g., phenylmethylsulfonyl fluoride, PMSF),
whereas others are tight binding (e.g., bovine
pancreatic trypsin inhibitor, BPTI, also known
as aprotinin) and some proteinase activity may
be recovered in later steps if inhibitor is no
longer present. This may cause problems if PIs
are omitted in later purification steps.
PMSF (1 mM), BPTI (1 M), and 4(2-aminoethyl)-benzenesulfonyl-fluoride hydrochloride (AEBSF or PEFA-BLOC, 1 mM)
are useful against serine proteinases. PMSF is
toxic and rapidly hydrolyzed, and should be
added to the extraction buffer just before tissue disruption.
EDTA, 1,10-phenanthroline, or 2, 2 bipyridyl (both at 1 to 10 mM) are used against
metalloproteinases, and trans-epoxysuccinylL-leucyl-amido-(4-guanidino)butane,
(E64,
10 M) is effective against cysteine proteinases. Antipain and leupeptin (both at
10 M) have activity against all serine
and cysteine classes. Pepstatin (10 M) is
effective against aspartic acid proteinases, but
may not be necessary at pH values near 7, as
aspartic proteinases generally function at low
pH values.
Commercial proteinase inhibitors (e.g.,
those provided in the form of tablets by Roche
and as solutions by Sigma) containing a cocktail of these inhibitors are available, tailored
for plants. These are a convenient way to add
a range of these inhibitors. In cases where it is
not desirable to add chelating agents, PIs without EDTA can be used. Note that PI tablets
are slow to dissolve, and so should be added
to buffer about 30 min before extracting the
tissue. Exercise caution, as some proteinase inhibitors are toxic.

Extraction of
Proteins from
Plant Tissues

Other special considerations

Filtration through Miracloth is effective, but
this material rips easily and is rather expensive. Nylon mesh is extremely strong, can be
washed and reused, and is readily available
from screen printing supply companies.
Whereas most plant proteins are not particularly heat- or acid-stable, some proteins can
be extracted successfully at low pH and/or at
high temperatures. For example, some small
proteinase inhibitors are stable at pH 3 (Ryan
et al., 2003), others at both pH 3 and 80 C for
6 min (Melville and Ryan, 1982).
Tissues with high lipid levels, such as seeds,
may need to be defatted before further purifica-

tion (Pastorello and Tranbaioli, 2001). Where

the protein of interest can withstand high
levels of organic solvent (e.g., as with proteinase inhibitors), the ground seeds can be defatted in acetone, the residue powder dried, and
the sample then extracted in aqueous buffer.
Enzyme inactivation is reduced by slowly
adding very cold acetone during extraction.
Rapid desalting of extracts
Large-scale extracts greater than 0.2 liters
(e.g., for protein purification) take longer
to process because centrifugation, filtering,
and other processes are slower. Consequently,
there can be a significant problem with
polyphenolics binding to proteins. By applying the centrifuged filtrate immediately to
a large (e.g., 40 10 cm) G-75 Superdex
or G-25 Sephadex column at a flow rate of
least 5 ml/min, various low-molecular-weight
polyphenols can be separated from proteins.
The column should be equilibrated with a
compatible buffer containing at least 100 mM
buffer to ensure that proteins do not bind to the
column.
For the preparation of small tissue samples
for crude assays, the extract can be desalted using small gel-filtration columns (e.g., PD-10
or NAP columns, Amersham Biosciences)
equilibrated with assay buffer or another compatible buffer. All steps can be done in microcentrifuge tubes in a microcentrifuge placed in
a cold room.
Often, there is a requirement to both protect the enzyme from modification in the extraction media and to rapidly concentrate the
protein for further work. The protein may be
concentrated by standard salt or organic solvent precipitation (Brovko and Zagranichnaya,
1998; UNIT 4.5). Alternatively, the extract can
be concentrated by dialysis against a volatile
buffer (e.g., ammonium carbonate), by using a filter-based concentrator (UNIT 4.4), or
by ion-exchange chromatography (UNIT 8.2).
However, concentration still may be slow and
may expose the protein to polyphenols and proteinases during these processes.
Tissue types
The types of tissue being extracted will affect the choice of method used and the yield of
protein. Leaves and roots have a fresh weight
of about 80% to 90% water, and can vary from
those that have few problem compounds except proteinases (e.g., in spinach) to those requiring the full spectrum of protectants (e.g.,
pine needles). Whereas protein concentration
in leaves can be high (1% to 4% of fresh

4.7.4
Supplement 38

Current Protocols in Protein Science

weight), the concentration in fruit and stem

tissue can be up to an order of magnitude lower,
and large extraction buffer ratios will lead to
very dilute protein concentrations in the extracts. Seeds, which are up to 90% dry weight
with correspondingly high protein concentrations, often contain high amounts of lipids,
which are difficult to separate from the aqueous extract.
Fruits are often acidic, with high levels of
polyphenolics, and need high buffer concentrations to maintain pH. The protein in this sort of
tissue is often very dilute and needs concentration. Some fruits (e.g., kiwifruit, papaya) are
also very high in cysteine proteinases.
Tissue culture cells and single-celled algae
can be difficult to break, and sonication, use
of a French press, or a cell walldigesting enzyme preparation (e.g., macerozyme) may be
necessary.
Vascular tissue needs to be separated from
surrounding material, although cambium peels
are sometimes possible. This can be done
by peeling the bark away from the stem and
scraping soft tissue from the inside of the
bark (phloem) or the outside of the stem
(xylem). Sometimes, specialized extracts
can be obtained; for example, it is very easy
to obtain large volumes of phloem sap from
cucurbits (Murray and Christeller, 1995). It is
more difficult to extract proteins from tissues
with secondary thickening of their cell walls,
such as woody tissues.
Tissues can be subfractionated to enrich for
the protein of interest (e.g., chloroplast or mitochondrial proteins) by density gradient fractionation of these organelles after tissue disruption. However, yields are usually low because short and gentle disruption methods are
needed to break cell walls without damaging
the membranes of the organelles. Membranes
can be isolated by differential centrifugation
(Rontein et al., 2003; Mika and Luthje, 2003)

before proteins are solubilized. Vacuoles, with

their single membrane, are particularly fragile and difficult to isolate. A common method
is to isolate vacuolar membranes (Yuasa and
Maeshima, 2000; Heyen et al., 2002). These
specialized methods are outside the scope of
this unit.
Each species may have unique problems.
Spinach is regarded as benign and easy to disrupt and is often used to make delicate active chloroplasts, whereas apples, tobacco, and
conifers are high in polyphenolics that crossreact with proteins. Arabidopsis is a model
plant species and is quite easy to work with and
extract proteins from (Che et al., 2003; Bonin
et al., 1997), as are potato (Geigenberger et al.,
1997) and tomato (Delmas et al., 2003).

Troubleshooting

See Table 4.7.1 for a listing of common

problems that may arise during extraction of
protein from plant tissues and suggestions for
how to overcome or avoid these problems;
these suggestions are elaborated upon in Critical Parameters.

Anticipated Results

Depending on the stability of the protein to

be extracted or purified, active protein should
be obtained easily from a wide variety of
plant materials including leaves of tree species,
woody tissue, and seeds (Table 4.7.2). Of
course, there are exceptions, and totally different approaches may need to be taken. For example, the most difficult tissue the authors have
encountered is the leaf of the New Zealand
beech tree (Nothofagus species), where the
best buffer was 50 mM borate, pH 7. For some
reason, this buffer complexed compounds in
the leaves that otherwise bound to proteins and
inactivated them (W. Laing and D. Stevenson,
unpub. observ.).

Table 4.7.1 Extraction Problems Common to Plants

Problem with plant tissue

Solutiona

Acidity (large acidic vacuoles)

Use high buffer amounts/weight tissue

Polyphenols

Use protective reagents

Oxidants

Use reducing reagents

Proteinases

Use proteinase inhibitors

Cell walls

Use efficient grinding techniques,

liquid nitrogen

High lipid content (e.g., seeds)

Defat using organic solvents

a See Critical Parameters for details.

Extraction,
Stabilization,
and
Concentration

4.7.5
Current Protocols in Protein Science

Supplement 38

Table 4.7.2 Examples of Proteins Extracted from Different Plants

Proteins assayed or
purified

Points illustrated by assay

Reference

Cell suspension Arabidopsis thaliana

cultures

GDP-mannose
3 ,5 -epimerase

Extraction and partial purification

of an enzyme from cell culture

Wolucka et al.
(2001)

Fruit cortex

Acid phosphatases

Illustrates some of the

Turner and
complications of a difficult tissue Plaxton (2001)

Fruit cortex and Actinidia deliciosa

seeds
(kiwifruit)

Phytocystatins

Extraction of robust proteinase

inhibitors, capable of withstanding
reversed-phase HPLC

Rassam and
Laing (2004)

Fruit phloem
exudate

Cucurbita maxima
(squash)

Aspartic proteinase
inhibitor

Extraction of a less robust

proteinase inhibitor

Christeller
et al. (1998)

Fruit tissues

Apple, kiwifruit,
avocado

Proteins for
two-dimensional
electrophoresis

A general method for analytic

separation of proteins from
difficult tissues

Barraclough
et al. (2004)

Leaf
chloroplasts

Spinacia oleracea
(spinach)

Glutamyl proteinase

Extraction involving isolation of

intact chloroplasts as the first step
in protein purification

Laing and
Christeller
(1997)

Leaves

Oryza sativa (rice)

Sucrose-1-phosphate
phosphohydolase

Extraction and purification of

an enzyme to homogeneity from
a monocot

Lunn et al.
(2000)

Seeds

Malus domestica
(apple)

Trypsin and papain

proteinase inhibitors

Extraction of robust proteinase

Ryan et al.
inhibitors, proteins capable of
(2003)
withstanding reversed-phase HPLC

Whole
seedlings

Arabidopsis thaliana

26S proteasome

Purification of a complex protein

from a model plant

Yang et al.
(2004)

Wood/xylem

Picea sitchensis
(Sitka spruce)

Laccase

Extraction of proteins from

woody tissue

McDougall
(2000)

Tissue

Plant

Musca cavendishii
(banana)

Time Considerations

In the authors laboratory, to purify proteins in large extracts, sequentially grinding

two 100 g lots of fresh tissue, extracting the
protein in extraction buffer, centrifuging, and
passing the extract through a large G-75 column can all be accomplished within a day,
with loading of the fractions containing the desired activity onto a Hitrap Q FF ion exchange
column the same evening. Alternatively, it is
reasonable to expect to be able to grind small
amounts of tissue (100 mg fresh weight), extract the frozen powder in buffer, centrifuge,
and apply to and elute from a NAP or PD-10
desalting column within 30 min. Where there
is a series of samples to extract, ten samples
can be extracted by two people within an hour.

Literature Cited
Extraction of
Proteins from
Plant Tissues

Barraclough, D., Obenland, D., Laing, W., and

Carroll, T. 2004. A method for quick and easy
two-dimensional electrophoresis of plant samples. Postharvest Biol. Technol. 32:175-181.

Bonin, C.P., Potter, I., Vanzin, G.F., and Reiter, W.D.

1997. The MUR1 gene of Arabidopsis thaliana
encodes an isoform of GDP-D-mannose-4,6dehydratase, catalyzing the first step in the de
novo synthesis of GDP-L-fucose. Proc. Natl.
Acad. Sci. U.S.A. 94:2085-2090.
Brovko, F.A. and Zagranichnaya, T.K. 1998. Separation of proteins from phenols in cereal leaf
extract by hydrophobic interaction: Ammonium sulfate fractionation. Plant Physiol. Bioch.
36:773-777.
Che, P., Weaver, L.M., Wurtele, E.S., and
Nikolau, B.J. 2003. The role of biotin in regulating 3-methylcrotonyl-coenzyme A carboxylase expression in Arabidopsis. Plant Physiol.
131:1479-1486.
Christeller, J.T., Laing, W.A., Ramsay, R.J.,
Cutfield, J., Cutfield, S., and Sullivan, P. 1998.
Purification and characterization of an aspartic
acid proteinase inhibitor from squash phloem exudate. Euro. J. Biochem. 254:160-167.
Delmas, F., Petit, J., Joub`es, J., Seveno, M.,
Paccalet, T., Hernould, M., Lerouge, P. Mouras,
A., and Chevalier, C. 2003. The gene expression
and enzyme activity of plant 3-deoxy-D-manno2-octulosonic acid-8-phosphate synthase are

4.7.6
Supplement 38

Current Protocols in Protein Science

preferentially associated with cell division in

a cell cycle-dependent manner. Plant Physiol.
133:348-360.
Geigenberger, P., Reimholz, R., Geiger, M., Merlo,
L., Canale, V., and Stitt, M. 1997. Regulation of
sucrose and starch metabolism in potato tubers
in response to short-term water deficit. Planta
201:502-518.
Heyen, B., Alsheikh, M.K., Smith, E.A., Torvik,
C.F., Seals, D.F., and Randall, S.K. 2002.
The calcium-binding activity of a vacuoleassociated, dehydrin-like protein is regulated by
phosphorylation. Plant Physiol. 130:675-687.
Laing, W.A. and Christeller, J.T. 1997. A plant
chloroplast glutamyl proteinase. Plant Physiol.
114:715-722.
Loomis, W.D. 1974. Overcoming problems of phenolics and quinones in the isolation of plant
enzymes and organelles. Methods Enzymol.
31:528-544.
Lunn, J.E., Ashton, A.R., Hatch, M.D., and Heldt,
H.W. 2000. Purification, molecular cloning,
and sequence analysis of sucrose-6F-phosphate
phosphohydrolase from plants. Proc. Natl. Acad.
Sci. U.S.A. 97:12914-12919.
Makkar, H.P.S., Blummel, M., and Becker, K. 1995.
Formation of complexes between polyvinyl
pyrrolidones or polyethylene glycols with tannins, and their implications in gas production
and true digestibility in in vitro techniques. Br.
J. Nutr. 73:897-913.
McDougall, G.J. 2000. A comparison of proteins
from the developing xylem of compression and
non-compression wood of branches of sitka
spruce (Picea sitchensis) reveals a differentially
expressed laccase. J. Exp. Bot. 349:1395-401.
Melville, J.C. and Ryan, C.A. 1972. Chymotrypsin
inhibitor I from potatoes: Large scale preparation and characterization of its subunit components. J. Biol. Chem. 247:3445-53.
Mika, A. and Luthje, S. 2003. Properties of guaiacol peroxidase activities isolated from corn root
plasma membranes. Plant Physiol. 132:14891498.
Murray, C. and Christeller, J.T. 1995. Purification
of a trypsin inhibitor (PFTI) from pumpkin fruit
phloem exudate and isolation of putative trypsin
and chymotrypsin inhibitor cDNA clones. Biol.
Chem. Hoppe Seyler 376:281-287.
Pastorello, E.A. and Trambaioli, C. 2001. Isolation
of food allergens. J. Chromatogr. B. Biomed. Sci.
Appl. 756:71-84.
Rassam, M. and Laing, W. 2003. Purification and
characterization of phytocystatins from kiwifruit
cortex and seeds. Phytochemistry 65:19-30.
Rontein, D., Wu, W.I., Voelker, D.R., and Hanson,
A.D. 2003. Mitochondrial phosphatidylserine
decarboxylase from higher plants: Functional
complementation in yeast, localization in plants,
and overexpression in Arabidopsis. Plant Physiol. 132:1678-1687.
Ryan, S.N., McManus, M.T., and Laing, W.A. 2003.
Identification and characterisation of proteinase

inhibitors and their genes from seeds of apple

(Malus domestica). J. Biochem. 134:31-42.
Schagger, H., and von Jagow, G, 1987. Tricinesodium dodecyl sulfate-polyacrylamide gel
electrophoresis for the separation of proteins in
the range from 1 to 100 kDa. Anal. Biochem.
166:368-379.
Schmalhausen, E.V., Pleten, A.P., and Muronetz,
V.I. 2003. Ascorbate-induced oxidation of
glyceraldehyde-3-phosphate dehydrogenase.
Biochem. Biophys. Res. Comm. 308:492-496.
Singh, B., Bhat, T.K., and Singh, B. 2003. Potential therapeutic applications of some antinutritional plant secondary metabolites. J. Agric.
Food Chem. 51:5579-5597.
Spencer, C.M., Cai, Y., Martin, R., Gaffney, S.H.,
Goulding, P.N., Magnolato, D., Lilley, T.H., and
Haslam, E. 1988. Polyphenol complexation
some thoughts and observations. Phytochemistry 27:2397-2409.
Turner, W.L. and Plaxton, W.C. 2001. Purification
and characterization of banana fruit acid phosphatase. Planta 214:243-249.
Wolucka, B.A., Persiau, G., Van Doorsselaere, J.,
Davey, M.W., Demol, H., Vandekerckhove, J.,
Van Montagu, M., Zabeau, M., and Boerjan,
W. 2001. Partial purification and identification
of GDP-mannose 3 ,5 -epimerase of Arabidopsis thaliana, a key enzyme of the plant vitamin C pathway. Proc. Natl. Acad. Sci. U.S.A.
9826:14843-14848.
Yang, P., Fu, H., Walker, J., Papa, C.M., Smalle, J.,
Ju, Y.M., and Vierstra, R.D. 2004. Purification
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Key References

Loomis, 1974. See above.

Describes in detail procedures to avoid the problems of phenolics.
Barraclough et al., 2004. See above.
Describes a robust method to process plant tissue
for two-dimensional electrophoresis.

Contributed by William Laing

The Horticultural and Food Research
Institute of New Zealand
Auckland, New Zealand
John Christeller
The Horticultural and Food Research
Institute of New Zealand
Palmerston North, New Zealand

Extraction,
Stabilization,
and
Concentration

4.7.7
Current Protocols in Protein Science

Supplement 38