Comparison of different protein
quantitation methods
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This application note compares the accuracy and reproducibility of
absolute protein quantitation of the Agilent 2100 bioanalyzer in combination
with the Protein 200 Plus assay to the batch-based methods
Lowry and Bradford as well as to denaturing gel-elctrophoresis (SDSPAGE).
The results showed that the Agilent 2100 bioanalyzer provides
fast and reliable absolute quantitation data comparable to the other
methods investigated.
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Determination of protein concentration
is a routine procedure in
many research laboratories. For
example, it is required to calculate
and monitor the protein yield after
various enrichment or purification
processes as well as to optimize
and standardize downstream
experiments such as protein-protein-
interaction studies.
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The Agilent 2100 bioanalyzer,
developed in collaboration with
Caliper Technologies Corp., provides
a compact lab-on-a-chip system
for the rapid and automated
analysis of proteins, DNA, RNA
and cells. Lab-on-a-chip technology
integrates multiple experimental
procedures, such as sample
handling, separation staining/
destaining, detection and analysis
in a single process. Together with
the Protein 200 Plus LabChip® kit
sizing and analysis of proteins
ranging in size from 14 to 200 kDa
is possible. In addition, it has the
ability to analyze relative quantitation,
based on internal standards
in each sample, and absolute
quantitation based on user-defined
calibration standards.
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This application note compares
the accuracy and reproducibility
of absolute quantitation determined
using the Protein 200 Plus
assay for the following three
methods: Lowry assay, Bradford
assay and denaturing gel electrophoresis
(SDS-PAGE) together
with a documentation and analysis
system.
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Experiment
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All proteins were purchased from
Sigma Aldrich GmbH (Taufkirchen,
Germany). Dulbecco's
PBS and distilled water were purchased
from Life Technologies
GmbH (Karlsruhe, Germany), -
mercaptoethanol was purchased
from Fluka (Buchs, Switzerland).
The Agilent 2100 bioanalyzer and
the Protein 200 Plus LabChip kit
were obtained from Agilent Technologies
Deutschland GmbH
(Waldbronn, Germany). All SDSPAGE
reagents and gels were purchased
from Invitrogen GmbH
(Karlsruhe, Germany).
Coomassie® Stain Solution and
Destain Solution were purchased
from BIO-RAD Laboratories
GmbH (Munich, Germany). The
digital camera and the imaging
software were purchased from
Kodak Digital Science, Eastman
Kodak Company (Rochester, NY,
USA). The Coomassie Plus Protein
Assay Reagent kit and the Modified
Lowry Protein Assay were
obtained from Pierce / Perbio
(Bonn, Germany). The Wallac 1420
Multilabel Counter was purchased
from Perkin Elmer Life Science
(Turku, Finland).
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Protein 200 Plus Assay
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The chip-based separations were
performed on the Agilent 2100 bioanalyzer
in combination with the
Protein 200 Plus LabChip kit and
the dedicated Protein 200 Plus
software assay. All chips were prepared
according to the protocol
provided with the Protein 200 Plus
LabChip kit. The kit includes 25
chips, spin filters and all reagents
needed for the experiments
including the Protein 200 Plus ladder
and the upper and lower marker premixed in the sample
buffer. The calibration feature of
the software was used to generate
a calibration curve with a linear
fit, the “unknown” protein concentrations
were determined automatically
by the software.
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SDS-PAGE
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Gel electrophoresis was performed
with 4-20 % Pre-Cast Tris-
Glycine Gels according to the
instructions provided by the manufacturer.
An equal volume Tris-
Glycine SDS Sample Buffer (2x)
was added to the samples, and
they were denatured for 5 minutes
at 95 °C before loading onto the
gel. The separation was performed
for approximately 120 minutes at
constant 125 Volts. Gels were
stained with Coomassie Stain
Solution for one hour and
destained overnight. A digital camera
was used for imaging and
analysis was performed with the
image analysis software. The
results were exported to
Microsoft® Excel and the data
points for the calibration curves
were fitted with a linear fit. The
resultant equation was used to calculate
the “unknown” concentrations
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Lowry and Bradford
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| Both batch-based methods were
performed using the Coomassie
Plus Protein Assay Reagent kit
and the Modified Lowry Protein
Assay respectively and a microwell
plate according to the
instructions of the manufacturer.
The absorbance was measured
with a fluorescence microwell
plate reader at 690 nm for Lowry
and 595 nm for Bradford measurements.
The results were exported
to Microsoft® Excel, the background signal was subtracted and
the data points for the calibration
curves were fitted with a second
order polynomial fit since no good
linear fit was obtained. The equation
that resulted was used to calculate
the “unknown” concentrations. |
Results and Discussion
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To study the differences in
absolute quantitation between the
different methods, three proteins
were tested: bovine serum albumin
(BSA), ovalbumin (OV) and
carbonic anhydrase (CA). The
quantitation accuracy and reproducibility
were determined for
each method. Five standards, 25,
100, 250, 500, 1000 and 2000 µg/ml,
were diluted from a 5000 µg/ml
stock solution for each of the individual
proteins. As reference for
the 5000 µg/ml stock solution, the
concentration supplied by the
manufacturer was used. In addition,
four so-called “unknown”
samples were diluted from the
same stock solutions, with concentrations
of 40, 200, 750 and
1250 µg/ml. To reduce variability
induced by sample preparation,
the same dilutions were used for
all four quantitation methods.
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Protein 200 Plus Assay
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The protein samples were analyzed
on the Agilent 2100 bioanalyzer
using the Protein 200 Plus
LabChip kit. This kit allows sizing
and quantitatation of 10 protein
samples in less than 45 minutes
including sample preparation with
a size resolution of approximately
10 % or better.
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The Agilent 2100 bioanalyzer software
provides two functionalities
for quantitation. The first possibility
is to determine the relative concentration
of the individual proteins.
This value is determined
automatically by the software
based on a one-point calibration
with the upper marker (myosin),
which is used as an internal quantitation
standard in every sample.
To determine the relative concentration,
the peak area of the
unknown sample is compared to
the peak area of the upper marker
with known concentration. The
inclusion of the upper marker in
each sample corrects for differences
in sample injection into the
separation channel and allows for reproducible quantitation. However,
due to some staining variability
between the upper marker and
the protein of interest, this value
is not as accurate as absolute
quantitation, using a pure sample
of the target protein as reference.
The Agilent 2100 bioanalyzer software
supports absolute quantitation,
which can be obtained by a
user-generated protein quantitation
calibration curve. This feature
was previously described1.
Figure 1 shows a screenshot of
the Agilent 2100 bioanalyzer software.
The data is displayed as gellike
image, as electropherograms
for each sample and in a tabular
format.
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The first six wells of the chip were
loaded with the carbonic anhydrase
standards with known concentrations
and the four
“unknown” concentrations were
loaded into wells seven through
ten. A calibration curve was
generated by the software, which
is depicted in figure 1. The
R-squared value of 0.998 clearly
shows the excellent linear behavior
of the protein assay. The result
table shows that the calibrated
concentration of 187.9 µg/ml is
greatly improved in comparison to
the relative concentration of 285.8
µg/ml. The target concentration in
this case was 200 µg/ml. All three
protein samples, BSA, OV and CA,
were analyzed using the same
experimental design. They were
run on six chips and three different
instruments—two chips per
instrument. Table 1 gives a summary,
showing that quantitation
accuracy was greatly improved
with the absolute quantitation feature
compared to the relative concentration.
The quantitation error
for CA was reduced from an average
error of 42 % to 6 %, from 28 %
to 11 % for OV and for BSA from
19 % to 15 %. The quantitation
reproducibility was approximately
10 % and comparable between
both quantitation methods.
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SDS-PAGE
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The same standards and samples
of the three proteins were also
separated on 4–20% gradient gels
for two hours at a constant voltage
of 125 V. For a better comparison
with the bioanalyzer the same
amount of starting material (4 µl)
was loaded in each gel well. The gels were stained with Coomassie
for one hour and destained
overnight. After that, a digital
image was taken (figure 2, top
panel) and the data was evaluated
with the gel electrophoresis analysis
software.
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The net intensity, which gives the
sum of the background-subtracted
pixel values in the band rectangle,
of the corresponding band from
the standards, was used to generate
a calibration curve with a linear
fit. Using a polynomial fit did
not improve data accuracy. The
lower panels of figure 2 shows the
averages of the different calibration
curves, which were calculated.
The displayed R-squared values
are between 0.95 and 0.98
indicating linear behavior, however
it is not as good as the linear fit
of the bioanalyzer. Table 2 shows
the quantitation results for the
four “unknown” target concentrations
determined with this
method. Relative standard deviations
(CVs) of around 13 % on
average were achieved for the
samples with concentrations
between 200 and 1250 µg/ml. The
quantitation of all protein samples
with a concentration of 40 µg/ml
resulted in CVs of 40 % or worse
and also showed a high deviation
from the target concentration. The
relatively high standard deviations
reflect the low staining and
destaining reproducibility from gel
to gel.
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The best results were obtained for
the highest target concentration of
1250 µg/ml showing good quantitation
reproducibility and an error
of 10 % or better. In contrast the 2100 bioanalyzer results are much
more consistent for all of the analyzed
target concentrations, providing
good and comparable quantitation
accuracy and reproducibility
across the tested concentration
range.
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Lowry
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Analysis with the Lowry method
was performed using the Pierce
modified Lowry protein assay
reagent. The microwell plate version
of the protocol was followed.
Therefore, a volume of 40 µl of each sample was consumed for
every measurement. Figure 3
shows the calibration curves
achieved with the six different
standards of each individual protein.
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Since non-linear color response
curves are typically obtained the
data points were fitted with a
second order polynomial fit. Both
R-squared values for BSA and OV
are 0.99 reflecting that the fit is
nearly perfect. In addition, standard
deviations are very narrow
indicating a good reproducibility
of the method for these proteins.
The calibration curve for carbonic
anhydrase is not optimal (R2 =
0.975) and also shows a relatively
high standard deviation compared
to the other two proteins tested.
This could be a hint that impurities
in the protein sample interfere
with the assay since it was not
observed with the other three
methods. The quantitation results
for the “unknowns” are shown in
table 3a.
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As expected from the calibration
curve the CVs for the carbonic
anhydrase samples are very high
ranging up to 55 % for the 40 µg/ml
sample. For BSA and OV the CV
values and the error values around
10 % are comparable to the bioanalyzer
quantitation data.
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Bradford
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The analysis with the Bradford
method was performed using the
Pierce Coomassie Plus protein
assay reagent kit. As for the Lowry
method, the microwell plate version
of the protocol was followed,
thus using 10 µl of each sample
for every measurement. The data points of the calibration curves
were fitted with a second order
polynomial fit. All R-squared values
were 0.99 (not shown). Standard
deviations are very tight indicating
a good reproducibility of
the method. Table 3b summarizes
the results of the quantitation of
the target concentration. Since the
working range specification for
this protocol given by the manufacturer
is only 100 to 1500 µg/ml,
the extremely high CV's and errors
for the lowest concentration of
40 ng/µl are explainable. All the
other values show comparable
CV's to the Lowry method and to
the data obtained with the 2100
bioanalyzer. However, the average
error of quantitation for the samples
within the specified concentration
range is around 28 % and therefore a factor of two to three
higher compared to the 2100 bioanalyzer
analysis and to the Lowry
method (figure 4).
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Comparison of the methods
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Summarizing the data obtained, all
four methods show similar behavior
in terms of absolute quantitation
accuracy. For better direct
comparison of the percent CV and
error values, a statistic is shown in
figure 4.
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With respect to reproducibility the
2100 bioanalyzer is comparable to
the batch-based methods Lowry
and Bradford and superior to SDSPAGE.
Also the percent error is
comparable to Lowry and even
better than Bradford and SDSPAGE.
However, the 2100 bioanalyzer has two big advantages
over Lowry and Bradford.
The batch-based methods allow
only for total protein quantitation,
whereas the 2100 bioanalyzer
quantitates each protein in a mixture.
In addition, the sample consumption
is 40 µl per sample for
Lowry and 10 µl using the Bradford
method, whereas the 2100
bioanalyzer only needs 4 µl of
sample. Analyzing protein mixtures
is also possible with SDSPAGE,
but this method is very
time-consuming, labor- intensive,
and produces hazardous waste in
larger quantities. Furthermore, the
researcher requires extra equipment
such as an imaging system in
order to evaluate the data. In contrast
the 2100 bioanalyzer offers a
speedy analysis of ten samples
within 45 minutes. No additional
staining or destaining is needed,
the data is evaluated automatically
by the software on the same
instrument and the hazardous
waste is significantly reduced.
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Conclusion
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The Agilent 2100 bioanalyzer in
combination with the Protein 200
Plus LabChip kit and the absolute
quantitation feature of the software
provides fast and reliable
absolute quantitation data. The
reproducibility and accuracy of
the method is comparable to
Lowry, Bradford and SDS-PAGE
measurements. However, the 2100
bioanalyzer displays several additional
advantages, which include
speed, automated detailed protein
analysis and significant reduction
of hazardous waste.
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