Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
SDS polyacrylamide gel electrophoresis (SDS PAGE), perhaps the most ubiquitous benchtop fractionation technique in use today, separates and orders proteins according to their apparent molecular weight. Yet in complex mixtures like cell lysates, many different proteins may have the same electrophoretic mobility and so a second, orthogonal technique often is required.
Two-dimensional gel electrophoresis (2DGE) provides that second level of separation. In 2DGE, proteins are separated first by their isoelectric point (the pH at which they are neutrally charged) and then by SDS PAGE, producing a 2D, visual readout like something painted by Jackson Pollock.
2DGE can individuate thousands of proteins on a single gel. Researchers typically use it to compare expression levels in different samples or to identify different protein isoforms, such as those differing by phosphorylation or glycosylation state, that may not be resolvable by size alone. Here’s what you need to know.
The first dimension
Sample preparation for isoelectric focusing (IEF) is a little trickier than for one-dimensional SDS PAGE. Salts, lipids and nucleic acids can interfere with IEF, as can protein solubility (proteins tend to aggregate near their isoelectric points). You may want to add chaotropes (e.g., urea and thiourea), nonionic or zwitterionic detergents (e.g., Triton X-100 or CHAPS) and reducing agents to the buffer—but make sure not to overheat it, lest the proteins react with the urea and become carbamylated, leading to artifactual results [1].
IEF traditionally was performed by loading the protein onto tube gels with a mixture of ampholytes (multiple-charged organic molecules) to form a pH gradient. A few brave souls, like those at Kendrick Laboratories, a Wisconsin-based contract research organization specializing in 2DGE, still pour their own gels (with a standard operating procedure for every step, notes president Nancy Kendrick).
But “pouring your own first dimension is Dark Ages technology,” says Jesse Guidry, director of the proteomics core facility at the Louisiana State University Health Sciences Center. “You used to fill the tube with acrylamide, and then once it polymerized you would squeeze the tube gel out of the tube. And then sometimes the gels would squeeze and stretch and break, and sometimes they would get turned around because you forgot which end was which. It was miserable.”
“Pretty much everyone exclusively now is using IPG strips,” says Sara Heitkamp, global proteomics product manager at Bio-Rad Laboratories. Immobilized pH gradient (IPG) strips are composed of polyacrylamide with the pH gradient cast into the gel, dried and affixed to a plastic backing. They are available as linear and nonlinear gradients, across various narrow and broad pH ranges, in several different lengths, from a variety of vendors.
The IPG strip can be loaded during rehydration, during which a large amount of evenly distributed protein can be laid on top of the strip, or protein can be added to a specific point on an already rehydrated strip. After current is applied, proteins migrate toward and remain at their isoelectric point (pI), so either loading approach will work.
A variety of horizontal and vertical electrophoresis systems are available to run IPG strips. Some vendors’ offerings are (semi-)interchangeable with their slab-gel apparatuses; others are specific to IEF, such as Bio-Rad’s PROTEAN® i12™ IEF system, which lets users independently control the voltage and current for each of the system’s 12 lanes. Users “can run completely separate sample types, different pH gradients, different protocols on the same IEF run,” says Heitkamp. “They can optimize their sample prep and conditions.”
The second dimension
After focusing, the proteins must be prepared for SDS PAGE. The proteins at their pIs are neutrally charged, so to be drawn into the second dimension they are first coated with SDS, typically in the presence of reducing and alkylating agents.
A second-dimension SDS PAGE gel is the same as a typical slab gel, except it has one very large well to physically accommodate the IPG strip, explains Heath Balcer, product manager for protein analysis at Life Technologies. Once in place, the strip is sealed into the well with a bead of agarose, and current is applied to cause the proteins to migrate.
After the second-dimension gel has been run, “there are a few different options,” says Heitkamp. Most people stain the gel with fluorescent, silver or Coomassie stain and capture the image on any of a number of standard gel-documentation systems. Combining the visual patterning generated by 2DGE with the sensitivity of Western blotting—using antibodies targeting specific modified protein forms, for example—can be an effective way to “drill down into the proteome,” says Kendrick.
Of course, many researchers go on to mass spectrometry (MS). Individual protein spots can be cut out of the 2D gel using anything from a single-edged razor blade, to dedicated spot-pickers like the OneTouch Plus from The Gel Company, to precision robotic spot cutters used in high-throughput applications. In this case, however, it’s important to plan ahead, because some stains—for example, most silver stains—and protocols may not be directly compatible with the MS workflow.
DIGE
For most users, 2DGE is a way to compare protein levels between different conditions, a workflow that necessarily requires comparing patterns across gels. Yet because of the inherent variability between 2D gels, comparing the thousands of spots from different samples can be a challenge. Software packages can help.
“Images for 2D gels don’t exactly overlay each other,” says Kendrick, who uses the stretching and warping functions of TotalLab’s SameSpots program to align the gels. “You only have to outline on one gel, and then the SameSpots algorithm propagates those outlines all across the gels, so all the spots are automatically matched. Then it can do background subtraction and automatically compare the volumes within the spot outlines.”
An alternative approach is 2D difference gel electrophoresis (DIGE), which eliminates gel-to-gel variability by running different samples and an internal control on the same gel. The technique calls for pre-labeling samples with size- and charged-matched fluorescent dyes (now marketed by GE Healthcare) [2]. After electrophoresis the gel is imaged, scanned and analyzed using GE’s DeCyder 2D software or another compatible package.
With the ascendancy of liquid chromatography/mass spectrometry workflows, the popularity of 2DGE certainly is not what it once was. Yet there is still much to be garnered from the comparatively low-cost, low-tech, two-dimensional visual separation that is 2DGE. If you need a quick-and-dirty look at the proteome, you might want to give the technique another look.
References
[1] Garfin, DE, “Two-dimensional gel electrophoresis: An overview,” Trends in Analytical Chemistry, 22:263-72, 2003. [PDF]
[2] Minden, JS, et al., “Difference gel electrophoresis,” Electrophoresis, 30:S156-S161, 2009. [PubMed ID: 19517495]
Image: Screenshot of TotalLab's SameSpots software.