An Automated Alternative to SDS-PAGE for Protein Analysis

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: labchip90@caliperls.com.

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