2D Gel Electrophoresis

by Jeffrey M. Perkel

In the world of proteomics, mass spectrometry-based techniques certainly get most of the glory. But the rumor of two-dimensional (2D) gel electrophoresis’ death has been greatly exaggerated.

“LC-MS is getting quite popular and widespread due to the quite rapid advance of mass-spec technology,” concedes Joe Hirano, product manager for 2D electrophoresis at GE Healthcare, which offers gel boxes, precast gels, reagents and imaging hardware to support the 2D gel application. “But there are still popular applications for 2D electrophoresis that have advantages over the general LC-MS approach.”

The 2D advantage

Post-translational modification studies: 2D gels, Hirano explains, separate intact proteins, whereas most mass spec-based approaches work with peptides. As a result, it can be difficult for researchers doing mass spec proteomics to determine, for instance, whether two distinct post-translational modifications were present in a protein simultaneously or are mutually exclusive. (The exception is “top-down” proteomics, a more technically demanding MS-based method in which intact proteins are separated prior to fragmentation.)

The big picture: 2D gels also provide a bird’s-eye view of the entire sample that MS-based techniques can’t match, says Katy McGirr, product manager at Bio-Rad Laboratories. In 2D gel electrophoresis, protein samples are resolved first by charge, in a step called isoelectric focusing (IEF), and then by size (as in standard SDS-PAGE). The result is an image in which potentially thousands of protein spots are resolved across the gel surface—images that can be studied and compared to see, for instance, how the proteome changes under certain conditions. “You can get a visual of what the entire sample looks like,” McGirr says.

Implementation:Finally, the two techniques differ in cost and infrastructure requirements. Although 2D gel equipment is relatively inexpensive, mass spectrometers represent a significant investment and require dedicated staff. Not everyone has access to a mass spectrometer or the expertise required to run it.

Of course, 2D gel electrophoresis isn’t new. The technique has been in use for decades. But that doesn’t mean there aren’t new developments. Whether researchers lack the equipment or the knowledge to do mass-spec proteomics or simply believe 2D electrophoresis fits their needs better, tools exist—and continue to be developed—to support their experiments.

Biomarker discovery with 2D gels

Tracey Madgett, a senior research fellow at Plymouth University, UK, has used 2D gel electrophoresis to identify potential biomarkers for Down Syndrome. As Madgett explains, the decision to adopt the electrophoretic approach stemmed from both scientific and practical considerations. First, others had already begun investigating Down Syndrome biomarkers with 2D gels, albeit using different sample types and conditions. But perhaps more importantly, her team had the 2D gel equipment in place and lacked the expertise needed for LC-MS. “That was another deciding factor,” she says.

Working with blood plasma from pregnant women in their first or second trimesters, carrying either a normal fetus or one with Down Syndrome, Madgett and her colleagues subjected 56 samples to 2D gel electrophoresis—in pairs. [1]

Traditionally, such an approach wouldn’t be feasible, as it isn’t possible to distinguish one sample from another in the gel. Therefore, each sample—say, treated cells or control cells—must be run, stained and analyzed independently. Yet gel-to-gel variability is commonplace, says Lena Jonsson, marketing program team leader at GE Healthcare. This makes it difficult to compare spot intensities across gels to identify differentially expressed proteins. “How do you know you can compare one spot on one gel with an equivalent spot on another gel?” Jonsson asks. In other words, is the protein at position x,y on one gel the same as the protein at the same position on another gel?

To circumvent that problem, GE Healthcare about a decade ago introduced a multiplexed technique called 2D DIGE, or differential in gel electrophoresis. In 2D DIGE, samples are labeled with different fluorescent dyes and then mixed and resolved on a single 2D gel, thereby eliminating the gel-to-gel variability between them. For instance, a control sample might be labeled with Cy3 and the treated sample with Cy5. A third sample, containing both control and treated sample (that is, all protein in the experiment), is labeled with Cy2 as an internal control. Because all the samples are treated identically during electrophoresis, researchers can more accurately identify, after the run, those proteins whose abundance differs between conditions; they simply collect three fluorescent images and compare them using data-analysis software.

Madgett and her team used 2D DIGE to study matched pairs of samples in their Down Syndrome biomarker analysis. The researchers first simplified their samples by eliminating the most abundant plasma proteins—albumin and immunoglobulin G—with the Qproteome Albumin/IgG Depletion Kit from Qiagen. They then resolved each pair on three 24-cm 2D gels, which differed in the pH range covered by the first dimension of the gel. In one gel, the proteins were resolved over the 4.5-5.5 pH range using a pre-cast GE Healthcare IEF gel called an immobilized pH gradient (IPG) strip. The second gel covered pH 5.3-6.5, and the third spanned pH 6-9. These narrow pH ranges offer higher resolution than do wider-range IPG strips (like those that cover pH 3-10), according to the researchers.

Their analysis, Madgett says, identified seven “promising leads” in second-trimester plasma that are more abundant in pregnant women carrying Down Syndrome fetuses than normal ones. The team identified those proteins by excising the spots and subjecting them to various mass-spectrometry techniques. Now, says Madgett, those leads need to be validated in larger pregnancy cohorts. “It’s complicated, but it’s very exciting in terms of getting the results in the end,” she says of the technique. “I would do it again.”

Variables to consider

Sample-preparation kits, IPG strips, IEF electrophoresis units and second dimension running units are all available from a wide variety of companies, including GE Healthcare, Bio-Rad, Life Technologies, Sigma-Aldrich and Serva, among others.

In broad strokes these devices are largely similar; it’s how they’re used that matters. And just as with one-dimensional gels, there are a number of variables to consider when running 2D gels.

Sample preparation: The first, says Hirano, is sample preparation. For instance, IEF is more sensitive to contaminants such as nucleic acids and lipids than are standard 1D SDS-PAGE gels, he says, “so getting rid of contaminants is good practice if you want to get nice, clean 2D gel runs.” GE Healthcare’s 2-D Clean-Up Kit efficiently separates proteins from contaminants such as salts, lipids, detergents and nucleic acids, says Helena Hedlund, the company’s product manager for protein sample preparation.

How big is your gel? Another variable, Hirano says, is the gel size. 2D gels can run from about 9 cm x 7 cm “mini gels” all the way up to about 24 cm x 20 cm, and IPG strips can run from about 7 cm to 24 cm in length. In general, he says, the larger the gel, the longer the run, but the better the resolution. On the flip side, says McGirr, larger gels are “more difficult” to handle. Bio-Rad supports both options, from the 4-gel Mini-PROTEAN® Tetra cell (7 cm IPG, 8.6 x 6.8 cm gels) to the 12-gel PROTEAN Plus Dodeca cell (18 to 24 cm IPG strips, 25 x 20.5 cm gels).

Choosing a pH range: Finally, there’s the IPG pH range, which can run the gamut from wide to narrow. For instance, Bio-Rad offers IPG strips covering “broad” (3-10), “narrow” (3-6, 5-8, 4-7) and “micro” (3.9-5.1, 4.7-5.9, 5.5-6.7, 6.3-8.3) pH ranges, for both sharper resolution and better separation of proteins. Yet according to McGirr, narrow ranges generally should be used only after a broader-range analysis has been completed. “If someone is working with a new or unknown sample, a broad pH range will give them an overview of the sample,” she says. “If they would like to get better resolution over a particular pI range [Is ‘pI’ correct? Or should it be ‘pH’?] or know the proteins they are interested in, narrow- and micro-range pH gradients would be recommended.”

Tips for optimal results

Researchers who are trying to home in on a specific region of the proteome, says McGirr, can either increase their gel size, narrow their IPG range or both. They can also fractionate their samples more extensively, for instance to look at specific subcellular compartments (nuclear, cytoplasmic or membrane, for instance) or subproteomes (e.g., phosphoproteins or glycoproteins), says Hirano. “With affinity enrichment [for instance, immunoprecipitation] you can get a much better identification rate, and you can also reduce the complexity of the sample to see the proteins you are most interested in,” he says. (Madgett used the Qiagen Qproteome kit to remove the most abundant plasma proteins from her analysis, thereby exposing the lower-abundance proteins she was more interested in.)

In any event, it’s a good idea to run samples more than once, which means researchers may find themselves needing to run multiple IPG strips simultaneously. Often, this is accomplished with an IEF system in which one power supply controls multiple electrophoresis lanes in serial.

There are several problems with that approach, says McGirr. First, it means all IPG strips being run at one time will theoretically be subjected to identical conditions (meaning multiple users with different needs cannot use the system simultaneously). Yet, says McGirr, that is precisely what doesn’t happen, at least if samples differ in their salt content, as the current is averaged over the entire tray. “Samples with higher conductivities will pull more current away from the strips with lower conductivities,” she explains, which can result in either over- or under-focusing.

The new PROTEAN® i12™ IEF system from Bio-Rad eliminates those two concerns with 12 independently controlled IEF lanes. “Each lane has its own power supply,” McGirr says, meaning it is now possible to run multiple focusing conditions simultaneously, optimize conditions and prevent samples from influencing one another.

Though she claims not to have had problems with her existing IEF system, Madgett is intrigued. “If different people in the lab are running different experiments, you can speed things up,” she says. “An IEF run tends to take overnight, and you are limited by how many overnights you have in a week.”

Reference

[1] Heywood, WE et al., “2D DIGE analysis of maternal plasma for potential biomarkers of Down Syndrome,” Proteome Sci, 9:56, 2011.

The image at the top of this page is from GE Healthcare Life Science's Deep Purple™ Total Protein Stain