Now that the Human Genome Project is finished,
the next important phase is understanding the
expression and function of proteins. Since many proteins
are key in cellular functions, thorough understanding
of protein expression and function in a
timely manner is essential for more efficient identification
of new targets for drug development.
Figure 1 - LabChip® 90 System.
Many researchers still use traditional methods
such as sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS-PAGE). This method is
time-consuming, labor-intensive, and can generate
a significant amount of hazardous waste. A
high-throughput, integrated instrument platform
has been developed that performs automated protein
sizing and relative quantitation. The
microfluidic assay is an automated alternative to
the manual SDS-PAGE analysis of proteins. The
LabChip® 90 System (Caliper Life Sciences, Inc.,
Mountain View, CA), shown in Figure 1, samples
directly from 96-well plates and integrates all
manual operations essential to protein analysis,
including staining, destaining, separation, detection,
and subsequent data analysis. While traditional
SDS-PAGE may take anywhere from 3 to 6
hr for electrophoretic separation and detection,
the LabChip 90 System protein assay accomplishes
all aspects of SDS-PAGE and additionally
provides quantitative data analysis of 96 samples
in approx. 1 hr.
Protein assay fundamentals
The protein assay is based on a microfluidic version of
SDS-PAGE. Proteins are denatured and coated with
SDS, which results in a net negative protein surface
charge that is approximately proportional to the
unfolded protein size. The SDS coating also provides
a hydrophobic environment for the fluorescent dye.
Instead of a cross-linked polyacrylamide gel, the system
uses microfluidic channels filled with an acrylamide
polymer solution, which is a sieving matrix for
separating the coated proteins according to their size.
Microfluidic chip function
Figure 2 - Detailed diagram of the protein chip (top-down view;
the sipper is located underneath the chip).
The protein chip performs several sequential functions
(Figure 2). First, it uses vacuum at well 1 to
aspirate approx. 170 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 1:1 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.
Figure 3 - Detailed view of the destain and detection region of the protein chip. The
image on the right is an actual photo of this region.
Next, the chip electrophoretically “loads” the
marker–protein mixture into the channel between
wells 3 and 8, where it crosses the separation channel.
A 20-pL sample plug is then 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. Each protein is stained with dye contained
in the gel and separated into distinct bands
with resolution comparable to a 4–20% gradient 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 3. In approx. 250 msec, diffusion
of free SDS micelles into the
SDS-free fluid results in breakup of
the micelles and a significant drop
in the background fluorescence.
Since the proteins are still coated
with SDS dye and retain their fluorescence,
the separated protein
bands are detected downstream of
the dilution point using laser-induced
fluorescence (LIF).
High-resolution
protein
electrophoresis
Figure 4 - Overlay of six electropherograms (identical samples), illustrating data reproducibility
of the system.
Figure 5 - The electropherogram on the far left is the actual data collected using the LabChip 90
System. The gel image on the left is the virtual gel generated from the system’s analysis of these
samples. The gel image on the right is an SDS-PAGE gel of the same samples. Sample buffer is 50
mM TrisCl, pH 7.5; 250 mM imidazole; 0.1% Tween-20. (Crude lysate samples and SDS-PAGE
data provided by Structural GenomiX Inc., San Diego, CA.)
Figure 6 - Reduced and unreduced forms of IgG antibody on the LabChip 90 System. The
reduced and unreduced forms are in alternate sample lanes in the gel image.
In Figure 4, six protein sample electropherograms
have been superimposed
to illustrate separation reproducibility.
The sizing range shown in
Figure 4 is from 14 to 200 kD. Peak 1 is the internal
marker dye and is used for normalization of sample size
and relative concentration. This automated normalization
of data ensures excellent data reproducibility.
Peak 2 is an SDS system peak that typically elutes at
approx. 6 kD. The software automatically excludes this
system peak and reports the migration time, peak
height, peak area, size, relative concentration, and
purity for each protein in a results table. Sizing and relative
concentration are calculated with respect to ladder
standards that are sipped at the beginning and end
of each row of 12 samples. Performance specifications
for the HT Protein Express assay are shown in Table 1.
Crude lysate and antibody
analysis
Crude cell lysate samples were analyzed using both
traditional SDS-PAGE and the LabChip 90 System
for comparison. The comparative data are shown in
Figure 5. The SDS-PAGE data show one protein
band at 48 kD, but the LabChip 90 System data
show two bands at the expected size. This suggests
that the LabChip 90 System provides better resolution
than the SDS-PAGE method for this protein
size range.
In addition to comparing crude lysates, both reduced
and unreduced forms of IgG antibody were analyzed
using the system (Figure 6). The results show that the
protein assay is able to consistently detect and characterize
both forms of the antibody. The reduced
forms of both heavy and light chains of the antibody
are very well separated and detected.
Conclusion
Automated sampling, staining, destaining, data analysis,
and data archiving features make the LabChip 90
System’s HT Protein Express Assay a powerful tool for
both low- and high-throughput laboratories requiring
high-quality protein analysis. The assay allows for more
efficient monitoring of the expression level of recombinant
proteins and purification processes and can also be
used for monitoring antibody
production. While traditional
SDS-PAGE data are
dependent on user variability
through staining, destaining,
and imaging steps, the
LabChip 90 System's use of
both an internal marker and
a standard ladder allows the
analysis of many samples
with a high level of sizing and relative concentration
reproducibility. Resolution, sensitivity, and dynamic
range are comparable or superior to SDS-PAGE, and
analysis is robust to varying salt concentrations and a
variety of buffers and additives. Individual sample results
are presented every 30 sec and complete analysis of a 96-well plate is achieved in approx. 1 hr. In addition, the
availability of both DNA and protein assays makes the
system an ideal solution for those conducting structural
genomics research.
The authors are with Caliper Life Sciences, Inc., 605 Fairchild
Dr., Mountain View, CA 94043, U.S.A.; tel.: 650-623-0700;
e-mail: [email protected].