C
H
A
P
T
E
R
17
Protein De termination
Martin Guttenberger
B. Bradford Assay
I. INTRODUCTION
From Bradford (1976): Coomassie brilliant blue G250 (Serva Blue G, Serva, Cat. No. 35050). The reagent
for this assay is available commercially from Bio-Rad
(Cat. No. 500-0006).
The protein content of tissues or samples can serve
a number of purposes: It can be a research topic of its
own (e.g., in nutritional studies; Hoffmann et al., 2002),
a loading control in gel electrophoresis (Unl/i et al.,
1997), or a reference quantity in biochemical (e.g.,
yields in protein purification) or physiological (e.g.,
specific activities of enzyme preparations; Guttenberger et al., 1994) investigations. In addition, with the
advent of proteomics, there is an increasing need for
protein quantitation in complex sample buffers containing deterg.ents and urea as potentially interfering
compounds (Unlii et al., 1997). In any case, care should
be taken to obtain correct results. This article focuses
on three techniques and outlines the specific pros
and cons.
C. Neuhoff Assay (Dot-Blot Assay)
From Guttenberger et al. (1991) and Neuhoff et al.
(1979): Ammonium sulfate for biochemical purposes
(Merck, Cat. No. 1.01211), benzoxanthene yellow
(Hoechst 2495, Merck Biosciences, Cat. No. 382057,
available upon request), cellulose acetate membranes
(Sartorius, Cat. No. SM 11200), glycine, and SDS (Serva,
Cat. Nos. 23390 and 20763, respectively). Commercially
available ammonium sulfate frequently contains substantial amounts of undefined UV-absorbing and fluorescing substances. These lead to more or less
yellowish solutions. Use only colourless solutions to
avoid possible interference in fluorometry.
Solutions are prepared from bidistilled water. Bovine
serum albumin (BSA, fraction V, Roche, Cat. No.
735086) is used as a standard protein. Ninety-six-well,
flat-bottomed polystyrene microtiter plates (Greiner,
Cat. No. 655101) are used for the photometric tests.
II. MATERIALS AND
INSTRUMENTATION
The following reagents are from the indicated
suppliers. All other reagents are of analytical grade
(Merck):
A. Lowry Assay
III. PROCEDURES
From Lowry et al. (1951): Folin-Ciocalteu phenol
reagent (Merck, Cat. No. 1.09001). A detergentcompatible modification of the Lowry assay is available as a kit (Bio-Rad 500-0116).
Cell Biology
With respect to convenience and speed, microplate
reader assays are described where appropriate. These
assays can be read easily in conventional instruments
131
Copyright 2006, Elsevier Science (USA).
All rights reserved.
13 2
PROTEINS
by employing microcuvettes or by scaling up the
volumes (fivefold).
The composition of the sample (extraction) buffer
requires thought with respect to the avoidance of artifactual alterations of the protein and to the compatibility with the intended experimental procedures. The
former requires strict control of adverse enzyme activities (especially proteases and phenol oxidases) and, in
the case of plant tissues, of interactions with secondary metabolites. A convenient, semiquantitative assay
for proteolytic activities allowing for the screening of
suitable inhibitors was described by Gallagher et al.
(1986). There is some uncertainty as to which assay
gives the most reliable results in combination with
extracts from plant tissues rich in phenolic substances.
The influence of such substances can never be predicted. It is therefore imperative to minimize interaction of these substances with protein in the course of
sample preparation. For a more detailed discussion of
this problem, see Guttenberger et al. (1994).
A frequent source of ambiguity is the use of the term
"soluble protein." Soluble as opposed to membranebound proteins stay in solution during centrifugation
for I h at 105,000g (Hjelmeland and Chrambach,
1984).
All assays described in this article quantitate protein
relative to a standard protein. The choice of the standard protein can markedly influence the result. This
requires special attention for proteins with a high
content of certain amino acids (e.g., aromatic, acidic, or
basic amino acids). For most accurate results, choose a
standard protein with similar amino acid composition
or, if not available, compare different assays and standard proteins. Alternatively, employ a modified Lowry
procedure that allows for absolute quantitation of
protein (Raghupathi and Diwan, 1994).
The most efficient way to prepare an exact dilution
series of the standard protein employs a handheld
dispenser (e.g., Eppendorf multipette). Typically a sixpoint series is pipetted according to Table I. In any
case, avoid a concentration gradient of the sample
buffer. Usually samples and standards may be kept at
-20~ for a couple of weeks. For longer storage intervals, keep at-80~
A. L o w r y Assay
See Lowry et al. (1951).
TABLE I
P i p e t t i n g S c h e m e for Preparation of
a Standard Dilution Series a
Concentration
Blank
0.2x
0.4x
0.6x
0.8x
1.0x
Water
Standard protein (2x)
Buffer (2x)
5
0
5
4
1
5
3
2
5
2
3
5
1
4
5
0
5
5
a To prepare i ml of each concentration, 1 volume corresponds
to 0.1 ml.
determinations), dissolve 20 g Na2CO3 in 1 litre
0.10 M NaOH. Keep at room temperature in tightly
closed screw-cap plastic bottles.
2. Reagent B: 0.5% CuSO4.5H20 in 1% sodium or potassium tartrate. To make 20 ml of reagent B, dissolve
0.1 g CuSO4-5H20in 20 ml 1% tartrate (0.2 g sodium
or potassium tartrate dissolved in 20 ml water).
Keep at room temperature.
3. Reagent C (alkaline copper solution): Mix 25 ml of
reagent A and 0.5 ml of reagent B. Prepare fresh
each day.
4. Reagent D (Folin-Ciocalteu phenol reagent): Dilute
with an equal volume of water just prior to use
Steps
1. Place 40 ~tl of sample (protein concentration 0.02I mg m1-1) or blank into cavities of a microplate or
into appropriate test tubes.
2. Add 200 ~tl of reagent C and mix. Allow to stand for
at least 10 min.
3. Add 20 ~tl of reagent D and mix immediately. Allow
to stand for 30 min or longer.
4. Read the samples in a microplate reader or any
other photometer at 750 nm.
Modifications
1. The sample volume may be raised to 140 ~tl when
samples are low in protein (0.02 mg ml q or less). In
this case, employ double-strength reagent C.
2. If samples have been dissolved in 0.5 M NaOH (recommended for resolubilization of acid precipitates),
omit N a O H from reagent A.
B. B r a d f o r d A s s a y
See Bradford (1976).
Solutions
Solutions
Note: For samples low in protein (0.02 mg m1-1 or
less), prepare reagents A and B at double strength.
1. Reagent A: 2% (w/v) sodium carbonate (Na2CO3) in
0.10 N NaOH. To make 1 litre of reagent A (5000
1. Protein reagent stock solution: 0.05% (w/v)
Coomassie brilliant blue G-250, 23.8% (v/v) ethanol,
42.5% (w/v) phosphoric acid. To make 200 ml of stock
solution (5000 determinations), dissolve 0.1 g Serva
PROTEINDETERMINATION
blue G in 50 m195% ethanol (denatured ethanol works
as well), add 100 ml 85% phosphoric acid, and make
up to 200 ml by adding water. The stock solution is
available commercially (Bio-Rad). Keep at 4~ The
reagent contains phosphoric acid and ethanol or
methanol. Handle with due care (especially when
employing a dispenser)!
2. Protein reagent: Prepare from the stock solution
by diluting in water (1:5). Filter immediately prior
to use.
Steps
1. Place 4 ~tl of sample (protein concentration 0.11 mg m1-1) or blank into cavities of a microplate or
into appropriate test tubes.
2. Add 200 ~tl of protein reagent and mix. Allow to
stand for at least 5 min.
3. Read the samples within I h in a microplate reader
or any other photometer at 595 nm.
Modifications
1. For improved linearity and sensitivity, compute the
ratio of the absorbances, 590 nm over 450 nm (Zor
and Selinger, 1996).
2. Microassay: For diluted samples (less than
0.1 mg ml-1), proceed as follows: Employ 200 ~tl of
sample and add 50 ~tl of protein reagent stock.
C. Dot.Blot Assay
See Guttenberger et al. (1991). Do not change the
chemistry of the membranes. Nitrocellulose will
dissolve in the staining solution; PVDF membranes
develop a strong background.
13 3
4. SDS stock: To make 30 ml of 10% (w/v) SDS stock
solution, dissolve 3 g SDS in approximately 20 ml of
water, stir, and make up to 30 ml (allow some time for
settling of foam). Keep at room temperature; it is stable
for at least 1 year.
5. Elution buffer: 0.25 M glycine-sulfuric acid buffer
(pH 3.6) and 0.02% (w/v) SDS. To prepare 1 litre, dissolve 18.8 g glycine in approximately 900 ml water and
add 15 ml of 0.5 M sulfuric acid. Slight deviations from
pH 3.6 are tolerable. Add 2 ml SDS stock and make up
to 1 litre. Keep at room temperature; it is stable for
months.
The following solutions are not needed for the
standard protocol.
6. Washing solution A: Saturated ammonium sulfate,
adjust to pH 7.0 with Tris. To make 1 litre, stir ammonium sulfate in warm water (do not heat excessively).
Let the solution cool to room temperature overnight
and titrate to pH 7.0 with a concentrated (approximately 2 M) solution of Tris (usually approximately
i ml is required). Keep at room temperature. As
ammonium sulfate tends to produce lumps in the
storage bottle it might be easier to weigh the entire
bottle, add some water, remove the resulting slurry,
and weigh the empty bottle again. To produce a saturated solution (53.1%, w / v ) , dissolve 760 g ammonium
sulfate in 1 litre water.
7. Washing solution B: Methanol/acetic acid/water
(50/10/40, v/v). To make i litre, mix 100 ml acetic acid
and 500 ml methanol; make up to 1 litre. Keep at 4~
8. Drying solution: 1-Butanol/methanol/acetic acid
(60/30/10, v/v). To make 0.1 litre, mix 10 ml acetic
acid, 30 ml methanol, and 60 ml butanol. Keep at 4~
use up to six times.
Steps
Solutions
1. Benzoxanthene stock: To prepare the stock solution
add i ml of water to 0.5 g of the fluorescent dye (as
supplied, weighing not necessary); keep at-20~ The
toxicity of benzoxanthene is not thoroughly studied, it
might be mutagenic!
2. Destaining solution: Methanol/acetic acid (90/10,
v/v). To make 1 litre, mix 100ml acetic acid and
900 ml methanol.
3. Staining solution: To obtain 100 ml, dilute 80 ~tl
benzoxanthene stock in 100 ml destaining solution. Be
sure to pour the destaining solution onto the stock
solution to prevent the latter from clotting. Keep
staining and destaining solutions in tightly closed
screw-cap bottles at 4~ in the dark. They are stable for
months and can be used repeatedly. Take due care in
handling the highly volatile solutions containing
methanol!
The dot-blot assay is a versatile tool; its different
modifications enable one to cope with almost every
potentially interfering substance. In the following
description the steps for all modifications are included.
1. Preparation of filter sheets (cellulose acetate
membrane). Handle the sheets with clean forceps and scissors, do not touch! Cut one corner to aid in orientation
during processing of the sheet. Mark the points of
sample application (see later). Mount the membrane
in such a way that the points of sample application are
not supported (otherwise a loss of protein due to
absorption through the membrane may be encountered). There are two different ways to achieve these
requirements.
a. For routine assays it is recommended to mount the
sheets in a special dot-blot apparatus (Fig. 1). Mark
dot areas by piercing the sheets through small holes
in the upper part of the device.
134
A
PROTEINS
I"
I
B
FIGURE 1 Dot-blot apparatus. (A) Top view. (B) Section along
the diagonal. The apparatus has not been drawn to scale. Dashed
lines indicate the position of the cellulose acetate membrane. Large
circles correspond to the application points, small ones to the
holes that are used for piercing the membrane (arrows in B), and
solid small ones to the position of the pins that hold together the
apparatus.
b. For occasional assays, mark the application points
by impressing a grid (approximately 1-cm edge
length) onto the filter surface (use a blunt blade and
a clean support, preferably a glass plate covering a
sheet of graph paper). Mount the sheets on a wire
grating (preferably made from stainless steel, fixation by means of adhesive tape is recommended; cut
off the taped areas prior to staining).
2. Apply samples (0.01-10 mg m1-1) to the membrane sheets in aliquots of 2 ~tl (piston pipettes are
highly recommended; well-rinsed capillary pipettes
may be used instead). Leave to dry for a couple of
minutes. Dilute samples may be assayed by applying
samples repeatedly (let the sample dry prior to the
next application).
3. Perform heat fixation. Note: This step is imperative
for samples containing SDS whereas it might prove deleterious to samples lacking SDS! Bake the dot-blot membranes on a clean glass plate for 10 min at 120~ (oven
or heating plate).
4. Remove interfering substances. Note: This step is
optional! Its use depends on the presence of potentially interfering substances (mainly carrier ampholytes, but also
peptides and the buffer PIPES). Remove interfering
substances prior to protein staining by vigorous
shaking in washing solution A (3 x 5 min), followed by
gentle agitation in washing solution B (3 x 2 min).
5. Stain and destain. Perform staining (10 min) and
destaining (5, 5, and 15 min) in closed trays (polyethylene food boxes work very well) on a laboratory
shaker at ambient temperature. For the last destaining
bath, employ fresh destaining solution; discard the
first destaining bath. The incubation times given here
represent the minimal time intervals needed. As long
as the vessels are closed tightly, each of these steps may
be delayed according to convenience (in case of the last
destaining bath, rinse in fresh destaining solution
before proceeding).
6. Dry the stained membrane sheets. To facilitate
cutting dot areas from the sheets, the following drying
step is recommended. Shake the membranes in drying
solution for exactly 2 min, mount them between two
clamps 1 (Fig. 2), and leave them to dry in a fume hood.
The dried sheets may be stored in the dark for later
analysis.
7. Elute. Prior to elution, cut the dots from the
membrane sheet. Perform elution (45 min in 2 ml of
elution buffer) in glass scintillation vials on a laboratory shaker at ambient temperature (bright illumination should be avoided). Dried sheets have to be
rewetted in destaining solution prior to immersion
in elution buffer. It is recommended to dispense the
destaining solution (25 ~tl) and the elution buffer with
appropriate repetitive devices (e.g., Eppendorf multipette and Brand dispensette, respectively).
8. Take
readings
in
a fluorometer
(e.g.,
Luminescence Spectrometer LS 50B; Perkin-Elmer;
Beaconsfield, UK) at 425 (excitation) and 475
(emission) nm.
Modification
Skip elution and take readings directly from the wet
membrane sheets (step 6) with a video documentation
system (e.g., DIANA, Raytest GmbH, Straubenhardt,
Germany; Hoffmann et al., 2002). Depending on the
choice of filters, there might be considerable deviation
from linearity.
IV. COMMENTS
With the exception of protein solutions, most stock
solutions have a long shelf life. Discard any stock solu1 Test for chemical resistance prior to first use: The edges of the
clamp can be protected by a piece of silicon tubing cut open along
one side.
PROTEIN DETERMINATION
Cons" High sensitivity to potentially interfering substances; least shelf life of the reagents employed.
Recommendation: E m p l o y where absolute protein
contents are of interest.
O"
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0
0
0
0
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0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
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13 5
7-
FIGURE 2 Membrane mounted for drying. Be sure to mount the
drying membranes between two clamps of sufficient size to prevent
distortion by uneven shrinkage. The weight of the lower clamp
should keep the membrane spread evenly.
tion that changed its original appearance (e.g., got
cloudy or discoloured).
Calculate standard curves according to the m e t h o d
of least squares. Appropriate algorithms are provided
with scientific calculators and most spreadsheet programs for personal computers. It is better to compute
standard curves employing single readings instead of
means. Be aware of the basic assumptions m a d e in
regression analysis. For additional reading on the
statistics of standard curves, compare Sokal and Rohlf
(1995).
A. Lowry Assay
Pros: The L o w r y assay exhibits the best accuracy
with regard to absolute protein concentrations due to
the chemical reaction with polypeptides. It is also
useful for the quantitation of oligopeptides. This
contrasts with the other two methods, which, as dyebinding assays, exhibit more variation d e p e n d i n g on
the different reactivity of the given proteins (standards
as well as samples).
B. Bradford Assay
Pros: The assay is widespread because of its
ease of performance (only one stable reagent is
needed, low sensitivity to potentially interfering substances, u n s u r p a s s e d rapidity), its sensitivity, and its
low cost.
Cons: High blank values, requires d u a l - w a v e l e n g t h
readings for linearity, and possibly rather high deviations from absolute protein values (depending on the
choice of standard protein).
Recommendation: E m p l o y where relative protein
contents are sufficient (in most cases such as electrophoresis) and where the assay shows no interference by sample constituents (compare Bradford,
1976).
C. Dot.Blot Assay
Pros: The dot-blot assay combines high sensitivity,
an extended range of linearity (20 ng to 20 btg), and
high tolerance to potentially interfering substances.
The sample is not used up during assay. Hence, it m a y
be reprobed 2 (Fig. 3) for immunological tests or detection of glycoproteins (Neuhoff et al., 1981).
Cons: More d e m a n d i n g and time-consuming than
the other assays and rather expensive (chemicals and
instrumentation).
Recommendation: E m p l o y where (1) the other
assays show interference, especially with complex
sample buffers used in one-dimensional 3 and twodimensional 4 electrophoresis; (2) the a m o u n t of sample
is limited a n d / o r reprobing of the dotted samples is
desirable; or (3) the mere detection of protein in
aliquots, e.g., from column chromatography, is n e e d e d
(spot 0.2-2 btl onto m e m b r a n e , process according to
2 Sheets containing single dot areas can be marked conveniently
by cutting the edges (Fig. 3, Neuhoff et al., 1979).
3 Sample buffer according to Laemmli (1970): 62.5 mM TrisHC1 (pH 6.8), 2% (w/v) SDS, 10% (v/v) glycerol, 5% (v/v) 2mercaptoethanol, and 0.001% (w/v) bromphenol blue. Range of the
assay: 0.04 to 10 mg m1-1, i.e., 80 ng to 20 btg in the test.
4 Sample (lysis) buffer according to O'Farrell (1975): 9.5 M urea,
2% (w/v) Nonidet P-40, 5% (v/v) 2-mercaptoethanol, and 2% (w/v)
carrier ampholytes. Standards are prepared by a stepwise dilution
of the BSA stock solution in a modified sample buffer lacking carrier
ampholytes. These are added from a doubly concentrated stock
solution (4%, w/v) in sample buffer. Range of the assay: 0.02 to
8 mg m1-1, i.e., 40 ng to 16 btg in the test.
13 6
PROTEINS
1
2
3
4
5
5. In microplates it is important to achieve uniform
menisci: Prick air bubbles with a thin wire and mix the
plates on a gyratory shaker.
6. Analysis of dilute samples by application of
larger sample volumes also increases the amount of
potentially interfering substances. Include appropriate
controls.
A. Lowry Assay
FIGURE 3 Usefulincision patterns employed for marking membrane sheets prior to reprobing. Additional patterns may be generated by combination.
standard protocol, prevent evaporation by covering
the destained membrane with a thin glass plate, view
under UV light).
V. PITFALLS
1. Solutions containing protein exhibit an altered
surface tension. Avoid foaming and pipette slowly and
steadily.
2. Extraction or precipitation steps to eliminate
interfering substances should be carefully controlled
for complete recovery of protein (Lowry et al., 1951).
The more demanding dot-blot assay frequently is a
good alternative because of a considerable gain of
convenience and accuracy with respect to a simplified
sample preparation.
3. Omission of known interfering buffer components from just those samples that are intended for
protein determination is strongly discouraged as
the solubility of proteins might be influenced
(carrier ampholytes, e.g., enhance solubilization of
membrane proteins in two-dimensional electrophoresis sample buffer; for references, see Guttenberger et
al., 1991).
4. In the case of photometers/fluorometers operating with filters (usually microplate readers), the correct
wavelength may not be available. Instead, a similar
wavelength may be employed [Lowry assay: 530800 nm, Bradford assay: 540-620 nm, dot-blot assay:
366-450 nm (excitation), 450-520 nm (emission)]. In
the case of fluorometry, allow for a sufficient wavelength interval between excitation and emission
(consult the operating instructions of your instrument). Be aware that considerable deviations from the
standard wavelengths will be at the expense of linearity and sensitivity.
1. Many reagents used commonly in protein
extraction interfere with this assay. The main groups
of interfering substances are reductants (e.g.,
sulfhydryl compounds such as mercaptoethanol,
reducing sugars such as glucose), chelating agents
(e.g., EDTA), amine derivatives (many common
buffering substances such as Tris), and detergents (e.g.,
Triton, SDS). A detailed list of interfering substances,
along with remedies and tolerable limits, is provided
by Petersen (1979).
2. Reagent D is not stable at a basic pH. Immediate
mixing after the addition of reagent D is imperative.
In microplates the use of a small plastic spatula is
convenient for this purpose (change or rinse between
samples).
3. The colour reaction takes about 80 min to come
to completion. Prior to this, reading of samples over an
extended period of time will give rise to experimental
error (more than 20%; Kirazov et al., 1993). Keep the
reading interval to a minimum. Alternatively, both
incubation steps can be cut to 3 min by raising the
incubation temperature to 37~ (Shakir et al., 1994). As
the time to reach thermal equilibration will depend on
the experimental setup, a test run in comparison to the
original method is recommended.
B. Bradford Assay
1. The commonly used standard protein BSA is
highly reactive in this dye-binding assay. As a consequence the protein content of the samples is underestimated. This systematic error does not matter in
comparative analyses but brings about wrong absolute
values. Bovine y-globulin is a preferable standard.
2. The standard curves are not strictly linear in the
original version of the assay. If the necessary equipment for the recommended dual-wavelength ratio
is not available, do not extend the range of standard
concentrations beyond one order of magnitude or do
not calculate standard curves by means of linear
regression.
3. Samples containing detergents (1% will interfere)
must be diluted (if possible) or precipitated (compare
Section V.2) prior to analysis.
PROTEINDETERMINATION
4. The p r o t e i n - d y e complex is insoluble a n d will
precipitate w i t h time (Marshall a n d Williams, 1992).
For highest accuracy, take readings w i t h i n an interval
b e t w e e n 5 a n d 20 m i n after a d d i t i o n of the reagent.
With crude extracts (e.g., from mycelia of certain
fungi), this interval m a y be considerably s h o r t e r m t o o
short to take m e a n i n g f u l readings. In this case, alter the
w a y of sample p r e p a r a t i o n or use another assay.
5. Plastic a n d glassware (especially quartz glass)
tend to b i n d dye. R e m o v e the resulting blue colour b y
one of the following procedures: (1) Rinse w i t h glassw a r e detergent (avoid strongly alkaline detergents
w i t h cuvettes; rinse t h o r o u g h l y to r e m o v e detergent
again), (2) rinse w i t h ethanol or methanol, or (3) soak
in 0.1 M HC1 (takes several hours).
C. Dot.Blot
Assay
1. Generally, it is imperative to p r e v e n t the m e m brane sheets from d r y i n g d u r i n g one of the transfer
steps (residual acetic acid will destroy the filter
matrix).
2. In case of highly variable results, inspection of
the stained filters (last destaining b a t h or dried) u n d e r
UV illumination m a y be helpful: B a c k g r o u n d staining
resulting from i m p r o p e r h a n d l i n g of the m e m b r a n e s
will be visible (do not use UV-irradiated m e m b r a n e s
for quantitative analyses).
3. After the w a s h i n g procedure, t h o r o u g h rinsing in
w a s h i n g solution B is imperative. A m m o n i u m sulfate
a c c u m u l a t i n g in the staining solution will interfere
w i t h the assay.
4. A l t h o u g h the dot-blot assay is extremely insensitive to potentially interfering substances, it is advisable to include a p p r o p r i a t e controls (at least b l a n k
buffer a n d buffer plus standard).
5. In the case of buffers containing detergent plus
carrier a m p h o l y t e s , the storage conditions a n d the
n u m b e r of f r e e z e - t h a w cycles m a y p r o v e i m p o r t a n t .
Use fresh solutions or r u n a p p r o p r i a t e controls.
6. If m e m b r a n e sheets t u r n t r a n s p a r e n t u p o n
drying, they have not been equilibrated p r o p e r l y in the
d r y i n g solution (keep in time: 2 min) or the d r y i n g
solution has been diluted by a c c u m u l a t i o n of destaining solution (do not reuse the d r y i n g solution too
often).
References
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle
of protein-dye binding. Anal. Biochem. 72, 248-254.
13 7
Gallagher, S. R., Carroll, E. J., Jr., and Leonard, R. T. (1986). A sensitive diffusion plate assay for screening inhibitors of protease
activity in plant cell fractions. Plant Physiol. 81, 869-874.
Guttenberger, M., Neuhoff, V., and Hampp, R. (1991). A dot-blot
assay for quantitation of nanogram amounts of protein in the
presence of carrier ampholytes and other possibly interfering
substances. Anal. Biochem. 196, 99-103.
Guttenberger, M., Schaeffer, C., and Hampp, R. (1994). Kinetic and
electrophoretic characterization of NADP dependent dehydrogenases from root tissues of Norway spruce (Picea abies [L.]
Karst.) employing a rapid one-step extraction procedure. Trees 8,
191-197.
Hjelmeland, L. M., and Chrambach, A. (1984). Solubilization of functional membrane proteins. Methods Enzymol. 104, 305-318.
Hoffmann, E. M., Muetzel, S., and Becker, K. (2002).A modified dotblot method of protein determination applied in the tanninprotein precipitation assay to facilitate the evaluation of tannin
activity in animal feeds. Br. J. Nutr. 87, 421-426.
Kirazov, L. P., Venkov, L. G., and Kirazov, E. P. (1993). Comparison
of the Lowry and the Bradford protein assays as applied for
protein estimation of membrane-containing fractions. Anal.
Biochem. 208, 44--48.
Laemmli, U. K. (1970). Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227, 680-685.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951).
Protein measurement with the Folin phenol reagent. J. Biol. Chem.
193, 265-275.
Marshall, T., and Williams, K. M. (1992). Coomassie blue protein
dye-binding assays measure formation of an insoluble proteindye complex. Anal. Biochem. 204, 107-109.
Neuhoff, V., Ewers, E., and Huether, G. (1981). Spot analysis for glycoprotein determination in the nanogram range. Hoppe-Seyler's Z.
Physiol. Chem. 362, 1427-1434.
Neuhoff, V., Philipp, K., Zimmer, H.-G., and Mesecke, S. (1979). A
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