Download Replacing Traditional SDS-PAGE and Bradford Techniques

Replacing Traditional SDS-PAGE and
Bradford Techniques in Monitoring of
Protein Purification Fractions Using
Agilent 5100 Automated Lab-on-a-Chip
Platform
Application
Authors
Paul Carter
GlaxoSmithKline Research and Development
Harlow, UK
Tanja Wulff
Agilent Technologies
Waldbronn, Germany
Abstract
The 5100 ALP, using the Protein 200 HT-2 assay, was used
to analyze all fractions from a purification process in a
faster and more automated way. A major advantage is
that the 5100 ALP provides both protein concentration and
purity simultaneously, eliminating the need for a Bradford
assay and SDS PAGE gels. Furthermore, the data is digital
and can be viewed as both a gel-like image as well as an
electropherogram. The electropherogram has the advantage that it is easier to see low level contaminants and
degradation products. The result-flagging feature in the
5100 expert software allows rapid identification of fractions meeting the researcher's predefined requirements
for purity and concentration. This time-saving capability
can easily indicate which fractions to pool, allowing the
user to more quickly purify expressed proteins for a range
of experiments.
Introduction
In the pharmaceutical industry, the demand for
pure proteins for a range of applications such as
structural biology, protein microarrays, target
validation studies, assay development, and highthroughput screening has increased to the point
where conventional, manual monitoring of protein
production and purification processes with
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis is rate limiting.
In order to shorten development times, expression
and purification experiments to optimize production of a particular protein are run in parallel,
resulting in a large number of samples to be
analyzed [1].
To enable crystallization of proteins to provide
structural information, the proteins need to be
soluble, of high purity, and highly concentrated. In
addition, the amount of a protein needs to be sufficient to conduct multiple experiments to identify
the optimum conditions for crystallization. The
determination of the three-dimensional structure
of a protein, in particular of its active site, can
serve as a basis for computer modeling, the design
of inhibitors, and an understanding of structureactivity relationships.
In this application note, we describe the purification process of a kinase domain which was used
for crystallization trials. The 5100 Automated
Lab-on-a-Chip Platform (ALP) was used to monitor
the entire process [2]. For comparison, an
SDS-PAGE analysis as well as a Bradford analysis
was also performed.
Materials and Methods
Purification method
The target protein, a kinase domain, was
expressed using the baculovirus system with a
directly fused 6xHis tag on the N-terminal.
The cells from 3.6 L of culture (3.6 × 109 cells) were
defrosted in 50 mL of buffer (50-mM Tris
pH 8.0, 200-mM NaCl, 50-mM imidazole) and disrupted by dounce homogenization, on ice. The
debris was removed by centrifugation (48000 g for
60 min at 4 °C). The supernatent was recovered
and loaded onto a 5-mL NiNTA Superflow column
(Qiagen), collecting five 10-mL fractions.
The column was washed after loading with
10 column volumes (50 mL) of buffer, collecting
ten 5-mL fractions, prior to elution using an imidazole gradient from 50 mM to 500 mM over
15-column volumes (75 mL), collecting thirty-three
2.5-mL fractions.
Fractions 20–32 from the NiNTA Superflow purification were pooled and concentrated to 4 mL by
ultra-filtration (Millipore, Ultrafree 15, 10 kDa
MWCO, 2500 g at 4 °C). The concentrated pool was
filtered (0.2 µm) and loaded onto a 100-mL
Superdex75 prep-grade column (GE Healthcare);
the column was eluted using 1.2-column volumes
of buffer (20 mM TRIS pH 8.0, 500 mM NaCl,
1-mM DTT) collecting 1-mL fractions.
SDS-PAGE analysis
Gel electrophoresis was performed with Cambrex
Duramide R 4%–20% Pre-Cast Tris-Glycine Gels.
Seven microliters of Tris-Glycine SDS Sample
Buffer (5x) was added to the samples (25 µL), and
they were denatured for 5 min at 95 °C before
loading onto the gel. The separation was performed for approximately 47 min at constant
250 V. Gels were stained with Coomassie Stain
Solution for 10 min and destained overnight. A
digital camera was used for imaging.
Bradford analysis
Bradford assays were performed in 96-well plates
using the Coomassie Plus Protein Assay Reagent
kit (Perbio) with BSA standards (Perbio). The
absorbance at 595 nm was measured using a
SpectraMax Plus 384 plate reader (Molecular
Devices) and the data analyzed using SoftMax Pro
(Molecular Devices) to calculate the protein
concentrations from the standard curve.
Protein 200 HT-2 assay using the 5100 ALP
The Agilent 5100 ALP was used to analyze the
purified protein samples. A Protein 200 HT-2
microfluidic chip was used according to the manu-
2
facturer's recommendations in conjunction with
the appropriate reagent kit. Unlike the Agilent
2100 bioanalyzer chip, this chip is reusable for
2000 samples and has external capillaries that
enable it to withdraw samples from a sealed titer
plate. The reagent kit comes complete with sample
buffer, protein standard ladder, storage buffer, and
ready-to-use reagent plates. Samples were prepared in Eppendorf PCR twin plates. A volume of
4-µL sample is dispensed to the well, followed by
2 µL of sample buffer. The plate is sealed with a
Remp plate sealer and heated to 95 °C for 5 min on
a PCR thermocycler. The plate is then cooled to
room temperature, the foil seal removed, and 24 µL
of deionized water added to each well, and mixed
thoroughly by pipetting. The plate is then resealed
with foil and is ready to run. One to twelve plates
can be placed in the racks of the 5100 ALP and
started for unattended measurement.
Results and Discussion
The target protein was expressed using the baculovirus system with a directly fused 6xHis tag on
the N-terminal and was purified using a two-step
purification process. The first purification step
performed was affinity chromatography using a
NiNTA Superflow column. Figure 1 illustrates how
the different fractions were collected. The flowthrough was collected as 10-mL fractions (1–5), the
wash was collected as 5-mL fractions (6–15), and
the elution was collected as 2.5-mL fractions
(16–48).
Each fraction was analyzed on the 5100 ALP using
the Protein 200 HT-2 assay. The principle of analysis used with the 5100 ALP is identical to that
used with the 2100 bioanalyzer [3, 4]. Proteins are
denatured with sample buffer containing dodecylsulfate and two internal standards (lower and
upper markers). These markers are used to correct
for sizing drift, which occurs from sample to
sample. In addition, the upper marker is used to
determine the relative concentration of proteins.
During the 5100 ALP run, all the data is stored digitally in an Oracle® database. While the instrument
produces electropherograms, data can also be
viewed as gel-like images. Detailed information like
size, concentration, and purity for each protein
peak can be viewed in peak tables. In addition, the
software calculates the total concentration of each
sample.
mAU
Conductivity
3000
2500
2000
Flow through
1500
Elution fractions
Wash fractions
1000
500
UV 280 nm
0
A1
A2
A3
A4
A5
0
1
Figure 1.
A6 A7 A8 A9 A10 A11 A12 B12 B11 B10 B8B7B6B5B4B3B2B1C1C2C3C4C5C6C7C8C9 C11 D12 D10 D8 D6 D4 D2
50
2
3
4
5
6
100
7 8 9 10 11 12 13 14 15 16
20
25
30
150
35
E1 Waste
mL
40
45
Fractions
Purification profile of the first purification step using a NiNTA Superflow column performed using
an AKTA purifier 100 chromatography system with a Frac950 fraction collector. Protein concentration as measured with UV 280 nm (blue trace) and conductivity (brown trace). Red marks indicate
protein fractions collected by the AKTA Purifier 100 system.
In Figure 2A, the gel-like image of the analyzed
fractions is shown. On the 5100 ALP, the protein is
running at 37 kDa, which is close to the expected
target size of 38.097 kDa, according to its amino
acid sequence.
Samples A1–A5 correspond to the flow through
fractions, A6–B3 correspond to the wash fractions,
and the elution fractions starting at sample B4.
The visual appearance of Figure 2A corresponds
very nicely to the UV 280-nm absorbance measurement in Figure 1.
A
B
Figure 2.
A) 5100 ALP analysis: gel-like image of the analyzed fractions from the NiNTA affinity chromatography
column. B) Corresponding SDS-PAGE analysis.
3
The highest concentration of the purified protein
can be found in samples B11–C2 (Figure 2A).
Figure 2B displays the analysis of the corresponding fractions with SDS-PAGE, stained with
Coomassie. Whereas the SDS-PAGE analysis of the
samples required two gels, the 5100 ALP is capable
of analyzing up to twelve 96-well plates in one
“job”. Therefore, samples can be compared more
easily and viewed in a single gel-view. Due to the
high reproducibility of the 5100 ALP, such comparisons can be performed with data generated within
a different “job”, on a different day, or even using a
different instrument. The software also calculates
the total protein concentration for each analyzed
sample. These results were compared against a traditional Bradford analysis and shown in Figure 3;
results from both methods match, both in trend
and in total concentration.
Fractions 20–32, corresponding to wells B8–C8 in
Figure 2A, were pooled and concentrated for the
second purification step, which was size exclusion
chromatography using a Superdex75 prep grade
column. Figure 4A shows the gel-like image of the
collected fractions. In addition, the fractions were
also analyzed on two SDS-PAGE gels (Figure 4B).
NiNTA fractions
ALP
Bradford
1400
Protein Concentration (µg/mL)
1200
1000
800
600
400
200
0
-200
1
Figure 3.
4
4
7
10
13
16
19
22
25
28
Fraction
31
34
37
40
43
46
Comparison of the total concentration of each column fraction determined by the 5100 ALP to a
Bradford assay.
49
A
B
Figure 4.
A) 5100 ALP analysis: gel-like image of the analyzed fractions eluted from the Superdex75 column.
B) Corresponding SDS-PAGE analysis.
The resolution of the SDS-PAGE gel is inferior to
the resolution of the 5100 ALP analysis. The analysis using the 5100 ALP provides a sizing resolution
of 10% difference in molecular weight range. Therefore, the 34 kDa protein, a degradation product, is
well resolved from the 37 kDa target protein in
sample D6.
Also, the superior linear dynamic range
(10–2000 µg/mL) of the 5100 ALP versus SDS-PAGE
helps to identify the difference between the
collected fractions. A more detailed view on this
double peak is shown in the electropherogram in
Figure 5A. The 37 kDa protein peak has a relative
concentration of 1093 µg/mL and a purity of 95.7%;
the 34 kDa degradation product has a relative concentration of 48.6 µg/mL and is an impurity of
4.3%. In contrast figure 5B shows a fraction (D1)
where the degradation product is not observed.
Such detailed information can also be used as
search criteria for large sets of samples and allows
the scientist to color label specific samples
according to the analytical results.
5
A
B
Figure 5.
6
A) Electropherogram view with sample information for sample D6. A 37 kDa protein peak and a degradation
product at 34 kDa is clearly resolved by the assay. B) Electropherogram view with sample information for pure
sample D1, where only a 37 kDa protein peak is visible.
This second level of data analysis, the so-called
result-flagging software feature, provides a fast
overview of the samples that were analyzed.
Figure 6 shows an example of result flagging.
The orange label reflects a sample containing a
37 kDa and a 34 kDa protein, and the yellow label
reflects samples which contain only a 37 kDa protein. All other samples that do not fulfill the search
criteria are labeled light blue. These result-flagging
rules can be much more complex and can be
adjusted to the needs and the project goals of the
user. It is therefore very convenient and allows the
user to focus immediately on the samples of interest. There is no need to look at all the analyses in
detail. The result-flagging rules can also be applied
across several “jobs” in the database, for example,
in order to monitor processes over time.
After completion of the purification project, a total
of 10.2 mg of pure target protein was recovered.
Figure 6.
Conclusion
In conclusion, the 5100 ALP using the Protein 200
HT-2 assay was used to analyze all the fractions
from a purification process in a faster and more
automated way. A major advantage is that the
5100 ALP provides both protein concentration and
purity simultaneously, eliminating the need for a
Bradford assay and SDS PAGE gels. Furthermore,
the data is digital and can be viewed as both a gellike image as well as an electropherogram. The
electropherogram has the advantage that it is
easier to see low-level contaminants and degradation products. The result-flagging feature in the
5100 expert software allows rapid identification of
fractions meeting the researcher’s predefined
requirements for purity and concentration. This
time-saving capability can easily indicate which
fractions to pool, allowing the user to more quickly
purify expressed proteins for a range of
experiments (5).
Sample plates displayed in the overview tab with an applied result-flagging: the orange label reflects
a sample containing a 37 kDa and a 34 kDa protein, the yellow label reflects samples which contain
a 37 kDa protein, and the light blue label reflects samples that do not meet either criteria. The
cross-hatched wells are protein ladder.
7
www.agilent.com/chem
References
1. Angela Davies, April Greene, Elke Lullau, and
W. Mark Abbott, “Optimisation and Evaluation
of a High-Throughput Mammalian Protein
Expression System”, (2005) Protein Expression
and Purification, 42 (1), 111-121.
2. Martin Greiner, Paul Carter, Bernhard Korn
and Dorothea Zink; “New Approach to Complete Automation in Sizing and Quantitation of
DNA and Proteins by the Automated Lab-on-aChip Platform from Agilent Technologies”.
(2004) Nature Methods, 1 (1), 87-89.
3. “Differences and Similarities Between the
Protein 200 Assay and SDS-PAGE”, Agilent
Technologies, publication 5988-3160EN
www.agilent.com/chem.
4. “The Protein 200 HT-2 Assay – How It Works
and How It Performs”, Agilent Technologies,
publication 5989-3636EN
www.agilent.com/chem.
5. Paul Hawtin, Ian Hardern, Mark Abbott,
Tanja Wulff, and Bill Wilson, “High Throughput
Protein Expression and Purification Analysis
Using the 5100 Automated Lab-on-a-Chip
Platform”, Agilent Technologies, publication
5989-3507EN
www.agilent.com/chem.
For More Information
For more information on our products and
services, visit our website at:
www.agilent.com/chem/5100ALP
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Information, descriptions, and specifications in this publication are subject to change
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© Agilent Technologies, Inc. 2005
Printed in the USA
September 14, 2005
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