Extraction of Proteins from Plant Tissues UNIT 4.

Proteins are extracted from plant tissues for a wide range of reasons, including to assay BASIC
an enzyme in a crude extract for physiological studies, to purify a protein in order to PROTOCOL
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 lignication. 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).

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 ltration
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 ne 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 4.7.1
Current Protocols in Protein Science (2004) 4.7.1-4.7.7
Copyright 
C 2004 by John Wiley & Sons, Inc.
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 ltered sample, centrifuge large-volume ex-
tracts >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, lter,
and centrifuge again. Combine the supernatants.
7. Depending on the purpose of the experiment, perform additional standard protein
purication 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 nely 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-puried 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
There are two main purposes for extract-
ing proteins from plant tissues: to assay pro-
tein levels or activity in a crude extract, or
to purify relatively large quantities of an en-
zyme or protein. The methods chosen thus
depend on purpose, tissue type, and enzyme
Extraction of or protein to be extracted. In this unit, vari-
Proteins from ous options are discussed for making appro-
Plant Tissues priate modications to cope with a particular
4.7.2
Supplement 38
situation. The more that is known about the
properties of the protein of interest, the eas-
ier it is to make choices regarding extraction
methods.
Generally, the better the health of the plant
tissue, the greater the activity that will be ex-
tracted (unless the target protein is a stress- or
disease-related protein). In addition, younger
plants and unstressed tissues often have fewer
interfering compounds.
Current Protocols in Protein Science
Critical Parameters
Extraction buffer
Buffer is required to maintain stability
of enzymes from both a pH and an ionic-
strength standpoint. Because plant cytoplasm
is around pH 7 and chloroplasts are at a
higher pH, it is generally optimal to main-
tain extracted enzymes at a similar, neutral
pH. However, extraction at pH > 7 is not rec-
ommended (see below). Alternative buffers to
MOPS include N-2-hydroxyethylpiperazine-
N -2-ethanesulfonic acid (HEPES), phos-
phate, 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, sufcient buffering ca-
pacity 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 conve-
nience of small volumes. Better extraction is
obtained with a higher ratio of buffer volume
to fresh tissue weight (e.g., 10 to 1). For large-
scale extractions, however, this often produces
too large a volume to work with in subsequent
steps unless the sample is rst concentrated.
An insufcient volume ratio of buffer risks
changes in the pH of the extract and modi-
cation 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 es-
sential to maintain the reduced state of free
sulfhydryl groups in enzymes and to reduce
oxidation of other components, such as phe-
nolics. Options include DTT (2 mM or more),
-mercaptoethanol (5 to 10 mM), or ascor-
bic acid (5 to 10 mM), and in some cir-
cumstances, stronger reducing agents such as
sodium dithionite (Melville and Ryan, 1972).
More than one reducing agent can be used
(e.g., DTT and -mercaptoethanol). Antioxi-
dants also reduce the activity of polyphenol ox-
idases that, in the presence of oxygen, rapidly
synthesize polyphenols from phenolics upon
tissue disruption. Polyphenol oxidase (PPO)
Current Protocols in Protein Science
inhibitors, such as sodium diethyldithiocar-
bamate (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 hydro-
gen peroxide, which can oxidize cysteine
(Schmalhausen et al., 2003). The iron chela-
tor 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 maxi-
mizing 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 bind-
ing 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 be-
low 7 (Makkar et al., 1995). PVP is useful
when making organelles or otherwise sepa-
rating samples from the medium (by centrifu-
gation or chromatography) early in the pro-
cedure. PVPP has the advantage that it is re-
moved from the medium at the ltration 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 fol-
lowing maceration. Cysteine proteinases are Extraction,
most widespread in plant tissues but metallo-, Stabilization,
serine, and aspartic proteinases have been and
Concentration
reported in different tissues. Furthermore, a
4.7.3
Supplement 38
 buffer pH of around 7 is conducive to cysteine
proteinase activity, as is the presence of an-
tioxidants. Some proteinase inhibitors are ir-
reversible and chemically modify their targets
(e.g., phenylmethylsulfonyl uoride, 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 purication steps.
PMSF (1 mM), BPTI (1 M), and 4-
(2-aminoethyl)-benzenesulfonyl-uoride hy-
drochloride (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 tis-
sue disruption.
EDTA, 1,10-phenanthroline, or 2, 2 -
bipyridyl (both at 1 to 10 mM) are used against
metalloproteinases, and trans-epoxysuccinyl-
L-leucyl-amido-(4-guanidino)butane, (E64,
10 M) is effective against cysteine pro-
teinases. 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 cock-
tail 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 with-
out 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 in-
hibitors are toxic.
Other special considerations
Filtration through Miracloth is effective, but
this material rips easily and is rather expen-
sive. 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 partic-
ularly 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
Extraction of 6 min (Melville and Ryan, 1982).
Proteins from Tissues with high lipid levels, such as seeds,
Plant Tissues
may need to be defatted before further purica-
4.7.4
Supplement 38
tion (Pastorello and Tranbaioli, 2001). Where
the protein of interest can withstand high
levels of organic solvent (e.g., as with pro-
teinase inhibitors), the ground seeds can be de-
fatted 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 purication) take longer
to process because centrifugation, ltering,
and other processes are slower. Consequently,
there can be a signicant problem with
polyphenolics binding to proteins. By apply-
ing the centrifuged ltrate immediately to
a large (e.g., 40 10 cm) G-75 Superdex
or G-25 Sephadex column at a ow 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 us-
ing small gel-ltration columns (e.g., PD-10
or NAP columns, Amersham Biosciences)
equilibrated with assay buffer or another com-
patible buffer. All steps can be done in micro-
centrifuge tubes in a microcentrifuge placed in
a cold room.
Often, there is a requirement to both pro-
tect the enzyme from modication in the ex-
traction media and to rapidly concentrate the
protein for further work. The protein may be
concentrated by standard salt or organic sol-
vent 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 us-
ing a lter-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 pro-
teinases during these processes.
Tissue types
The types of tissue being extracted will af-
fect 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 ex-
cept proteinases (e.g., in spinach) to those re-
quiring the full spectrum of protectants (e.g.,
pine needles). Whereas protein concentration
in leaves can be high (1% to 4% of fresh
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 ex-
tracts. Seeds, which are up to 90% dry weight
with correspondingly high protein concentra-
tions, often contain high amounts of lipids,
which are difcult to separate from the aque-
ous extract.
Fruits are often acidic, with high levels of
polyphenolics, and need high buffer concentra-
tions to maintain pH. The protein in this sort of
tissue is often very dilute and needs concentra-
tion. Some fruits (e.g., kiwifruit, papaya) are
also very high in cysteine proteinases.
Tissue culture cells and single-celled algae
can be difcult to break, and sonication, use
of a French press, or a cell walldigesting en-
zyme 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 difcult 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 mi-
tochondrial proteins) by density gradient frac-
tionation of these organelles after tissue dis-
ruption. However, yields are usually low be-
cause 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)
Table 4.7.1 Extraction Problems Common to Plants
Problem with plant tissue
Acidity (large acidic vacuoles)
Polyphenols
Oxidants
Proteinases
Cell walls
High lipid content (e.g., seeds)
a See Critical Parameters for details.
Current Protocols in Protein Science
before proteins are solubilized. Vacuoles, with
their single membrane, are particularly frag-
ile and difcult 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 dis-
rupt and is often used to make delicate ac-
tive chloroplasts, whereas apples, tobacco, and
conifers are high in polyphenolics that cross-
react 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 Crit-
ical Parameters.
Anticipated Results
Depending on the stability of the protein to
be extracted or puried, 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 differ-
ent approaches may need to be taken. For ex-
ample, the most difcult 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.).
Solutiona
Use high buffer amounts/weight tissue
Use protective reagents
Use reducing reagents
Use proteinase inhibitors
Use efcient grinding techniques,
liquid nitrogen Extraction,
Stabilization,
Defat using organic solvents and
Concentration
4.7.5
Supplement 38
Table 4.7.2 Examples of Proteins Extracted from Different Plants

Proteins assayed or
Tissue Plant Points illustrated by assay Reference
puried

Cell suspension Arabidopsis thaliana GDP-mannose Extraction and partial purication Wolucka et al.
cultures 3 ,5 -epimerase of an enzyme from cell culture (2001)
Fruit cortex Musca cavendishii Acid phosphatases Illustrates some of the Turner and
(banana) complications of a difcult tissue Plaxton (2001)
Fruit cortex and Actinidia deliciosa Phytocystatins Extraction of robust proteinase Rassam and
seeds (kiwifruit) inhibitors, capable of withstanding Laing (2004)
reversed-phase HPLC
Fruit phloem Cucurbita maxima Aspartic proteinase Extraction of a less robust Christeller
exudate (squash) inhibitor proteinase inhibitor et al. (1998)
Fruit tissues Apple, kiwifruit, Proteins for A general method for analytic Barraclough
avocado two-dimensional separation of proteins from et al. (2004)
electrophoresis difcult tissues
Leaf Spinacia oleracea Glutamyl proteinase Extraction involving isolation of Laing and
chloroplasts (spinach) intact chloroplasts as the rst step Christeller
in protein purication (1997)
Leaves Oryza sativa (rice) Sucrose-1-phosphate Extraction and purication of Lunn et al.
phosphohydolase an enzyme to homogeneity from (2000)
a monocot
Seeds Malus domestica Trypsin and papain Extraction of robust proteinase Ryan et al.
(apple) proteinase inhibitors inhibitors, proteins capable of (2003)
withstanding reversed-phase HPLC
Whole Arabidopsis thaliana 26S proteasome Purication of a complex protein Yang et al.
seedlings from a model plant (2004)
Wood/xylem Picea sitchensis Laccase Extraction of proteins from McDougall
(Sitka spruce) woody tissue (2000)

Time Considerations
In the authors laboratory, to purify pro-
teins 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 col-
umn can all be accomplished within a day,
with loading of the fractions containing the de-
sired 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), ex-
tract 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.
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Wolucka, B.A., Persiau, G., Van Doorsselaere, J.,
Davey, M.W., Demol, H., Vandekerckhove, J.,
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W. 2001. Partial purication and identication
of GDP-mannose 3 ,5 -epimerase of Arabidop-
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9826:14843-14848.
Yang, P., Fu, H., Walker, J., Papa, C.M., Smalle, J.,
Ju, Y.M., and Vierstra, R.D. 2004. Purication
of the Arabidopsis 26S proteasome: Biochemi-
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Yuasa, K. and Maeshima, M. 2000. Purication,
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Key References
Loomis, 1974. See above.
Describes in detail procedures to avoid the prob-
lems 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
Supplement 38