Determining protein function

Automated electrophoretic sizing and concentration analysis of proteins means efficient identification

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.

Many researchers still use traditional methods such as SDS-polyacrylamide gel electrophoresis (SDS-PAGE). This method is time-consuming, labour-intensive, and can generate a significant amount of hazardous waste. Caliper Life Sciences has developed a high-throughput, integrated instrument platform that performs automated protein sizing and relative quantitation. This microfluidic assay is an automated alternative to the manual SDS-PAGE analysis of proteins. The LabChip 90 System 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 the traditional SDS-PAGE may take anywhere from 3 to 6 hours 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 approximately 1 hour.

Microfluidic devices offer a number of important benefits for chemical analysis, including rapid separation, less reagent consumption, automation, integration of multiple sequential steps, and potential for high sample throughput. These benefits have been successfully demonstrated in the miniaturisation of capillary electrophoresis (CE), and agarose gel electrophoresis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and for separation of DNA fragments and proteins, respectively. The main difference between microchip format and CE is the ability to inject a very small plug of sample (picolitres). On a microfluidic chip this is achieved by having a channel network where a sample load channel crosses the separation channel forming a cross. The small volume of the sample plug is determined by the size of the channel widths, typically on the order of 30-80µm.  The time scale for diffusion-limited separation is substantially reduced when introducing a sample plug having 10s of µm in dimension. The injection of the well-defined plug at the channel intersection is typically achieved by controlling the voltages at the four terminal ends of the channels. An efficient electrophoretic separation of the injected plug is achieved in channels with length of less than 15mm that contain a sieving matrix.

Protein assay fundamentals
The LabChip 90 System 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 LabChip 90 System uses microfluidic channels filled with an acrylamide polymer solution, which is a sieving matrix for separating the coated proteins according to their size.

The LabChip 90 system chip

The LabChip 90 System protein chip performs several sequential functions referenced in Figure 1. First, it uses vacuum at Well 1 to aspirate approximately 170nL 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.

Next, the chip electrophoretically loads the marker-protein mixture into the channel between Wells 3 and 8, where it crosses the separation channel. A 20pL 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% SDS-PAGE gel. Protein de-staining is accomplished using a dilution step achieved by electrokinetically flowing SDS-free ions into the separation channel at the de-stain 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. Since the proteins are still 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).

High resolution protein electrophoresis
In Figure 3, six protein sample electropherograms have been superimposed to illustrate separation reproducibility. The sizing range shown in Figure 3 is from 14 to 200kDa. Peak 1 is the internal marker dye and is used for normalisation of sample size and relative concentration. This automated normalisation of data ensures excellent data reproducibility. Peak 2 is an SDS system peak that typically elutes at approximately 6kDa. The software automatically excludes this system peak and reports the migration time, peak height, peak area, size and relative concentration 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.

Crude lysate and antibody analysis
Crude cell lysate samples were analysed using both traditional SDS-PAGE and the LabChip 90 System for comparison. The comparative data are shown in Figure 4. The SDS-PAGE data show one protein band at 48kDa, but the LabChip 90 System data show two bands at the expected size. This suggests that the LabChip 90 provides better resolution than the SDS-PAGE method for this protein size range.

Figure 4. Actual data collected using LabcChip 90 system on left, virtual gel on right.

In addition to comparing crude lysates, both reduced and unreduced forms of IgG antibody were analysed using the LabChip 90 System (Figure 5). The results show that the LabChip 90 System protein assay is able to consistently detect and characterise both forms of the antibody. The reduced forms of both heavy and light chains of the antibody are very well separated and detected.

Figure 5. Reduced and unreduced forms of the IgG antibody on LabChip 90 system.

Conclusions
Automated sampling, staining, de-staining, 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, de-staining, 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 seconds and complete analysis of a 96-well plate is achieved in approximately 1 hour. In addition, the availability of both DNA and protein assays makes the LabChip 90 System an ideal solution for those conducting structural genomics research.

By Jackie Richards, Product Manager, Caliper Life Sciences
enquiry number 05104