Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
If there’s one thing you can count on finding in today’s molecular-biology labs, it’s a protein gel electrophoresis rig. Researchers use protein gels to resolve proteins by size and/or—in the case of 2D gels—charge, whether in preparation for Western blotting or mass spectrometry, or simply to screen protein expression by eye.
But as with DNA gels, it’s not enough to simply separate proteins in a gel; they also must be stained. As it turns out, there are several ways to make proteins visible. Which approach to use is a function of sensitivity requirements, available hardware and downstream application.
Basic staining approaches
There are three primary classes of stains researchers can use to treat their everyday protein gels: Coomassie, silver and fluorescent. According to Jeffrey Turner, senior research and development manager for protein technologies and assays at Sigma-Aldrich, choosing between those options basically comes down to “sensitivity vs. speed.”
Coomassie is probably the most commonly used stain, Turner says, and as a visual, colorimetric (bright-blue) dye, it requires no special equipment. But it also offers the lowest sensitivity, with a detection limit of about 10 to 100 ng protein, according to Ning Liu, business development manager at Bio-Rad Laboratories.
Companies typically offer multiple formulations of the dye, which vary in ease of use, overall staining time and buffer and destaining requirements. Some, for instance, require fixation in an alcohol/acid wash, whereas others use pure water. Expedeon’s InstantBlue™ reagent (also available from Sigma-Aldrich) is added directly to the gel post-electrophoresis, no fixation or destaining required, and staining is complete in about 15 minutes. Bio-Rad Laboratories’ QC Colloidal Coomassie Stain protocol, on the other hand, recommends a longer procedure to meet industrial quality control standards, including a 15-minute gel-fixation step in 40% ethanol/10% acetic acid prior to staining for between one and 20 hours, followed by one to three hours of destaining in water.
Researchers can automate Coomassie staining with Thermo Fisher Scientific’s newly released Pierce™ Power Stainer. According to Emily Goplen, global product manager, this device can stain and destain two gels in a single step. “While traditional staining will take multiple hours to overnight, the Power Stainer completes the staining and destaining process in about 10 minutes and works with either precast [or] homemade SDS-PAGE gels,” she says.
At the other extreme is silver staining, a visual method with sensitivity of about 100 pg to 250 pg (0.1 ng to 0.25 ng) per band. The protocol has no destaining step, says Liu, but “it is a long procedure”—two hours or so—“and quite finicky.” Plus, adds Turner, the protocol involves several toxic chemicals, such as formaldehyde and silver, and typically is carried out in a fume hood. As a result, says Liu, “people usually stay away from that procedure,” unless sensitivity is of paramount importance.
In the middle, sensitivity-wise, are fluorescent dyes, which can detect proteins in the single-digit nanogram range, provided you have access to a compatible gel documentation system or transilluminator.
Like Coomassie and silver staining, fluorescent staining is used post-electrophoresis, and it often requires both fixation and destaining. But that is not always the case. For instance, says Liu, though SYPRO Ruby requires a destaining step, Oriole and Flamingo gel stains do not, as the free dyes are not fluorescent. “The [dye] itself cannot be excited by the laser,” he explains, “only when it is bound by the protein. That is why you don’t need to wash it [out].”
Supplementing these options, specialty reagents also exist. For instance, Thermo Fisher Scientific offers Pro-Q® fluorescent stains specifically for glycosylated and phosphorylated proteins, as well as the InVision™ His-tag In-gel Stain and Lumio™ Green Detection Kit for nanogram-level visualization of tagged fusion proteins.
Pre-electrophoresis stains
Some companies offer staining solutions that eliminate post-electrophoretic washes and staining. For instance, Bio-Rad’s Stain-Free™ pre-cast gels come preloaded with a tryptophan-binding “trihalo” fluorescent dye. After electrophoresis, exposure of the gel to ultraviolet light activates the dye, causing it to bind covalently to protein. According to Liu, sensitivity “is on par with Coomassie.”
Another option is the Amersham™ WB system from GE Healthcare Life Sciences business. With this system, samples are covalently coupled to the Cy™5 fluorophore prior to electrophoresis in a 10- or 30-minute reaction. Destaining is not necessary—free dye runs off the gel with the dye front—and sensitivity is in the 50- to 100-pg range, with a minimum dynamic range of three orders of magnitude, says Åsa Hagner McWhirter, a senior scientist at GE Healthcare Life Sciences. “You don’t even have to disassemble the gel cassette” to see the gel pattern, McWhirter says; the system automatically images the gel directly at the end of electrophoresis and analyzes the results.
Both Liu and McWhirter note that one significant advantage of the pre-electrophoretic staining approach is that the dyes are covalently attached to the separated proteins. Thus, it is possible to visualize them in subsequent steps, especially in a Western blot, and to normalize Western target signals using total protein signal rather than selected “housekeeping”-protein levels. (Western membranes also can be stained to obtain total protein signal by using such reagents as Coomassie blue, Ponceau S and Thermo Fisher Scientific’s Pierce™ Reversible Protein Stains.)
“This approach obviously eliminates two big concerns that people have about the housekeeping-protein loading control,” Liu says. First, housekeeping-protein levels can sometimes change across experimental conditions. And they may also exist at levels outside the blot’s linear dynamic range, complicating quantification. Indeed, he notes that the Journal of Biological Chemistry recently updated its author guidelines to reflect that fact. “Normalization of signal intensity to total-protein loading (assessed by staining membranes using Coomassie blue, Ponceau S or other protein stains) is preferred,” the guidelines now state. “’House-keeping’ proteins should not be used for normalization without evidence that experimental manipulations do not affect their expression.”
Pre-labeling also substantially increases gel-to-gel reproducibility, says McWhirter, as wash and stain steps can be difficult to control precisely. “The CVs [coefficients of variance] between the reactions that you do in the tubes are much lower than incubating gels in different solutions using trays,” she notes. Indeed, she claims that in one in-house analysis of 14 identical samples, CVs for protein Cy5 signals averaged about 5%. “That’s basically as good as pipetting errors.”
Final considerations
Whichever stain you choose, one key consideration, in addition to sensitivity, speed and hardware requirements, is what you plan to do with the gel downstream. For instance, researchers don’t typically stain gels with Coomassie blue prior to blotting, as that dye often requires a fixation step that can trap the protein in the gel; instead, they either run a second identical gel in parallel and stain that, or stain the blot after transfer. Similarly, dyes that covalently attach to proteins may potentially interfere with antibody binding or mass spectrometric analysis.
Ultimately, there is no one-size-fits-all solution, Goplen says. Coomassie is most researchers’ go-to stain, all agree. But when special circumstances arise, isn’t it nice to know there are plenty of additional options available?