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.
For decades, Polyacrylamide gel electrophoresis (PAGE) has been the method of choice for separating proteins. In PAGE’s simplest form, proteins are loaded onto a lattice-like slab gel that is then subjected to an electric field, causing the proteins to be pulled through the gel. Larger chains take longer than shorter ones to migrate through the lattice, resulting in a ladder of distinct bands of different mass proteins.
“All protein gels are similar in that they’re a matrix of polymerized acrylamide,” says Jonathan Kohn, group leader in R&D with Bio-Rad Laboratories. “It’s the buffering system that differs from gel type to gel type.”
By varying that buffering system—broadly defined to include the proportion of acrylamide (and bis-acrylamide, a cross-linking agent) as well as salts, reducing agents, detergents and dyes included in the formulations—proteins large and small, native and denatured, modified or not, can be separated, visualized and used in a host of applications.
Westerns
Much of protein electrophoresis is performed as a precursor to a Western blotting, an extremely sensitive way to visualize proteins after they have been separated. Proteins on the gel are generally transferred to a nitrocellulose or PVDF membrane and probed (directly or indirectly) with labeled antibodies that recognize specific proteins of interest. This has traditionally been a long and laborious process, and it’s in the researcher’s own best interest to make sure both the electrophoresis and the transfer were successful before proceeding to time-consuming, potentially expensive blotting steps.
Because the Coomassie blue, silver stain and others stains used to visualize proteins on an acrylamide gel can interfere with the downstream Western blot, researchers often run two identical gels, one for staining and one for transferring to a membrane. The stains bind to all the proteins found on the gel, giving an indication of their migration patterns (and thus relative sizes). Relative quantities of protein often can be estimated from the stain, as well; however, caution is advised, as proteins do not always take up the stains evenly.
One way to avoid this duplication of effort is offered by Bio-Rad, in the form of its stain-free chemistry. “The gel contains a component that binds to their protein and can be seen in one of our stain free-enabled [fluorescent] imagers. The researcher[s] can run their gel, put it on the imager directly, take a look at it and see what’s on their gel,” says Kate Smith, marketing manager in the company’s Laboratory Separations Division. “Now they have the opportunity to check the separation on their gels before they then go on to blotting,” without the multi-hour process of staining and de-staining with Coomassie.
Yet, partly because of advances in gel transfer—both Bio-Rad and Life Technologies now offer ways to transfer proteins from a gel to a membrane blot in as little as three minutes, for example – and partly because of the ease of reversible membrane staining, Life Technologies’ customers haven’t requested such a thing, “because so few people are actually doing Coomassie stains anymore. They just are immediately transferring to the membrane,” says Jennifer Cannon, senior development marketing manager at Life Technologies. “Nobody is really stopping at that step to say, ‘Did my gel work?’ because they’ve got a pre-stained standard. Then when they transfer they can run a Ponceau S stain and look at the total stained membrane.”
Mass spec and other apps
Other downstream PAGE applications are compatible with, and to a large extent demand, staining the gel. Many researchers use PAGE to prepare proteins for introduction into a mass spectrometer, for example. There are a variety of gel stains and staining kits that are compatible with mass spectrometry (MS)—including Coomassie and its relatives—with a selection of desirable properties. Thermo Scientific, for example, offers stains that do not require fixing and that can be de-stained using water; some have protocols that can be performed in considerably less than an hour; some are completely reversible; some are colorimetric; and some can detect protein down to sub-nanogram level, notes Priya Rangaraj, market segment manager for Thermo Scientific protein detection products.
After the separated proteins are visualized, bands can be excised from the gel. They can then be protease digested in-gel and introduced into an MS protocol. The technique has become popular in the past five to 10 years. “Gel-C-MS is usually the most biologist-friendly because it’s familiar—people know where the protein will run, so . . . you can see if the gel band is there, potentially, before you cut it out,” says Aaron Sin, marketing manager for protein biology at Sigma-Aldrich, who points out it’s an easy way for a biologist to interface with an MS lab.
“And a lot of times it’s also used because the gel can cut down on a lot of the salt that you have in your sample. Once you go in the gel, the salt doesn’t matter anymore,” Sin adds. “It saves a step of desalting.”
PAGE is used to prepare samples for other purposes, as well. “I’ve used it myself by cutting off bands from a gel, eluting the protein and using that as an immunogen to generate antibodies,” says Rangaraj. People also use the excised bands for sequencing and other purposes.
Gels may be run as a quality control in the protein-purification process, to check the purity or yield. It’s also common for PAGE to be used to compare protein expression levels, for example, between cell lines, batches or processes.
Going native
A large majority of PAGE is done in the presence of the anionic detergent SDS, which denatures the proteins and imparts a roughly even distribution of charge proportional to the protein’s mass. Most often, a reducing agent such as β-mercaptoethanol (BME, 2-ME) also is added to the sample buffer. Thus, when the proteins are subjected to an electric field, they migrate solely according to their masses.
With native gels, in contrast, no detergent (and typically no reducing agent) is used. “If you’re running a native gel, your intention is not to denature the protein, [but rather] to gain information not only about its size but about its shape and charge,” Kohn explains. “That may be because you want to gain information about its active conformation or native structure for some subsequent activity assay or structural analysis or what have you.”
Native gels also are used to query very large proteins and protein complexes, adds Sin.
If you’re planning to purchase a precast gel, it’s important to check with the manufacturer as to whether it is compatible with native applications. Bio-Rad’s offerings can be used for either denaturing or native gels, for example. On the other hand, Life Technologies sells gels specifically for native applications, and the company’s website warns that although its other gels “do not contain SDS, they are formulated for denaturing gel electrophoresis applications only.”
Buy it or pour it
The majority of researchers purchase precast gels rather than cast their own. And there are those labs that do both, says Smith. “A lot of times, labs will use both precast and hand-cast gels, using precast gels when it comes to generating publication-quality images. So they realize that the image quality will be much higher and the reproducibility is higher with a precast gel,” she says.
On the flip side, “if I’m doing some . . . quick checks, and I’m not too worried if the gel smiles, or if I get poor resolution, or it doesn’t look pretty, then I’ll pour my own gel,” points out Cannon. The researcher will typically pour a few gels at a time, store them cold and use them within a week because of the short shelf life of hand-cast gels.
Longer shelf life—up to a year, in many cases—reproducibility, convenience and efficiency of the workflow are not the only reasons a researcher may choose to purchase rather than pour.
Most precast gels contain a gradient (rather than a fixed percentage) of polyacrylamide; such gels are difficult to hand-cast, usually involving specialized equipment. Labs tend to stock up on gels, says Cannon, and because there is no price differential between gradient and fixed-percentage gels, purchasing gradient gels gives them the ability to query different size ranges of proteins when the need arises.
Chemistry
Furthermore, some of the newer, more specialized chemistries are either proprietary (like the stain-free) or require some skill to achieve.
“For researchers that really want the best type of resolution—the sharpest bands, the best sensitivity, the least amount of degradation—they tend to use the bis/tris chemistry. That requires a different type of buffer than the traditional Laemmli buffers, as well,” notes Cannon. “The bis/tris chemistries are based on a MOPS or a MES buffer type. You have to be a pretty good chemical wizard to make up the bis/tris gradient gel and pour it yourself, and the MOPS and MES buffers are rather difficult to get into solution.”
Bis/tris isn’t best suited for all separations, though. Tricine gels are best for very low molecular weight proteins, for example, and tris/acetate may be preferred for very large proteins. And glycosylated proteins resolve better on a tris/glycine gel. (But tris/glycine gels are more likely to give protein modification that will be seen as an artifact in downstream MS applications.)
There are also new variants on the traditional tris/glycine chemistry, such as Bio-Rad’s TGX, which is designed for faster running times and Western transfers.
Decisions, decisions
Kohn advises that “the decision factor within that very large range of sample types and protein sizes mostly devolves down to choice of the appropriate percentage to generate a separation pattern that allows you to really zoom in on a particular portion of the mass range.” If you’re looking at a single protein or narrow range, choose a fixed-percent gel. Otherwise use the appropriate gradient.
Beyond that, certain applications—quick QC, publication-quality imaging, gel transfer or band excision, for example—may dictate the type of chemistry to use and whether to pour your own gel or purchase one ready-made.
The image at the top of the page is from Life Technologies.