| Jun
1, 2005 |
| By:
Sherri
Biondi, Adrian
Winoto, Jim
Mikkelsen, Bahram
Fathollahi |
| Pharmaceutical
Discovery |
|
Introduction Presently, a need
exists in protein laboratories for rapid sample analysis in a format that
enables quantitative decision-making. As experimentation becomes more
complex and broader in scope, whether it involves testing expression in
multiple cell lines, growth conditions or constructs, it becomes
infeasible to compare, analyze and make decisions based on data from gel
images alone. Automated processes and direct data reporting enable life
scientists to view and manipulate protein sizing, concentration and purity
data in order to make the most informed decisions possible in
high-throughput experimentation. Questions related to purity, process
optimization, expression level and solubility must be answered quickly and
accurately in order to move target proteins on to functional assays,
crystallography or final production in a much more efficient and timely
manner.
Although SDS-PAGE is the traditional method for protein analysis, data
can be variable and results are qualitative. Parts of the gel process can
be automated, but a significant amount of manual interaction still is
required. Microfluidic assays quickly are becoming the preferred solution
for protein analysis in laboratories requiring more information in an
expedient manner. The LabChip 90 system performs automatic sampling from a
microtiterplate followed by electrophoresis, data analysis and reporting.
Because sample loading, injection and separation can be precisely
controlled on the microfluidic chip, analytical data is highly
reproducible.
The Protein Express assay generates quantitative sizing, concentration
and purity data as each sample is processed. Sensitivity is comparable to
a mid-range colloidal coomassie stain, with a large dynamic range. The
Protein Express assay encompasses a wide variety of comparable gel
concentrations, which allows a broader resolution range to be achieved.
Quality of data is higher, laboratory throughput is drastically increased
and time to answer is reduced significantly.
Table I: Protein Express Assay
Specifications
|
Protein Assay Fundamentals The
Protein Express assay is a microfluidic version of SDS-PAGE, where each
step of the slab gel process – sample loading, electrophoresis,
staining, destaining and detection – is integrated into a microfluidic
device. Quantitative sizing, relative concentration and purity results are
reported for each sample. Sample analysis takes approximately 35 seconds,
and a full 96-well plate can be analyzed in just over an hour. Results can
be viewed in three formats: a gel-like display, an electropherogram and a
tabulated results table. Detailed sample information can be imported into
the software for tracking purposes. Protein data also can be exported for
presentations, data archiving or database submission.
Microfluidic Chip Function
Preparation of the protein chip and samples can be completed in
approximately 20 minutes. The samples are heat denatured in a high
concentration of SDS. The SDS coats the protein, which results in a net
negative protein surface charge that enables electrophoretic separation.
The protein chip is prepared by pressure priming the microfluidic channels
with gel-dye and destain solutions. The gel-dye solution serves as both a
sieving matrix for the separation of the proteins and a staining solution.
Once the protein chip is primed, a marker solution is pipetted onto the
chip. Both the chip and the protein plate then are loaded onto the
machine, and the assay is started.
Figure 1. A detailed diagram of the
LabChip 90 protein chip. This is a top-down view; the sipper
extends out underneath the chip.
|
The protein chip performs several sequential functions, as referenced in
Figure 1. First, it uses vacuum applied to well 1 to aspirate
approximately 130 nL of sample from the well plate through a capillary
sipper and into the microfluidic channels of the chip. During this step,
the sample is diluted 2:1 with a marker solution, which is simultaneously
drawn from well 4. This marker is subsequently used as a reference for
migration time and determination of relative concentration of samples.
Next, the chip electrophoretically
"loads" the marker-protein mixture into the channel between
wells 3 and 8, across the separation channel. A 40 pL sample plug then is
electrophoretically injected into the separation channel. A potential is
applied between wells 7 and 10, which causes the individual proteins in
the sample to migrate up the separation channel.
Figure 2. A detailed view of the
destain and detection region of the LabChip 90 protein chip. The
image on the right is an actual photo of this region.
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Each protein is stained with dye
contained in the gel and separated into distinct bands with resolution
comparable to a 4–20% SDS-PAGE gel. Protein destaining is accomplished
using a dilution step achieved by electrokinetically flowing SDS-free ions
into the separation channel at the destain intersection. This causes the
dye-SDS-protein fluid stream to focus, as shown in Figure 2. In
approximately 250 milliseconds, diffusion of free SDS micelles into the
SDS-free fluid results in breakup of the micelles and a significant drop
in the background fluorescence. SDS micelles bound to the protein remain
intact. Since the proteins still are coated with SDS-dye and retain their
fluorescence, the separated protein bands are detected downstream of the
dilution point by using laser induced fluorescence (LIF). Free solution
dye molecules are not detected because they are only fluorescent in the
hydrophobic environment of the SDS micelles.
Protein Expression Monitoring Using the
Protein Express Assay There are
many applications for protein sizing and quantitation using the LabChip 90
system. These applications can include the monitoring of protein
expression and solubility, analysis of column fractions and purified
proteins and antibody QC, among others.
In protein expression, cells are
modified specifically to over-express proteins of interest. These cells
are then lysed, followed by an extraction and purification of soluble
protein. Protein analysis is commonly performed on the whole-cell lysate
to determine the degree of protein expression, on the supernatant to
determine expressed protein solubility and then on the partially purified
fractions to determine purity. Further processing and analysis of the
insoluble cell material often are conducted to confirm expression levels.
Not all expressed proteins are seen in the soluble component of the cell
lysate due to precipitation. Analysis of the insoluble cell material
provides further verification of protein expression levels and is
performed to potentially avoid inaccurate conclusions drawn from initial
data.
Figure 3. Whole-cell lysate, soluble
and insoluble lysate fractions and Ni bead purified sample data
generated for expressed E. coli proteins.
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The data shown in Figure 3 represents
the various stages of the expression process. Escherichia coli cells were
used to over express a ~40 kDa protein. Cells were lysed using sonication
and subsequently centrifuged to obtain soluble proteins. A magnetic Ni
bead-based affinity purification was conducted on the soluble proteins.
Further processing of insoluble material was also conducted.
Conclusion
Automation of the SDS-PAGE process allows scientists to spend valuable
time on experimentation and research, rather than processing slab gels.
The time-consuming and labor-intensive manual slab gel process can
sometimes generate variability in data that may be unreliable over time
and across experiments. Using the LabChip 90 system's Protein Express
assay, high-quality sizing and quantitation data is quickly presented,
allowing more accurate decisions to be made much sooner in the expression
process. Both high- and low-throughput laboratories can take advantage of
the system's automated analysis, as workflow flexibility permits anywhere
from just a few samples to multiple plates to be analyzed throughout the
day as needed. Run times of approximately one hour can result in more than
a three-fold increase in laboratory throughput. Compatibility with
microplates makes upstream automation possible by permitting automation of
the entire sample preparation and analysis process, both of which are the
most common bottlenecks faced by protein laboratories today. The digital
data format allows results to be compared between experiments and shared
easily between multiple groups at multiple sites, and simplifies database
population. The LabChip 90 system's Protein Express assay can be a
powerful tool for protein expression, purification, production and
engineering groups requiring efficient analysis of lysates, column
fractions, purified proteins, and antibodies.
Caliper Life Sciences
68 Elm Street
Hopkinton, MA 01748-1668 USA
www.caliperls.com
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