Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Separating proteins in a polyacrylamide gel according to their molecular weights (or more accurately, their electrophoretic mobilities) is a standard technique scientists use for studying and potentially isolating proteins. Upon completion of SDS-PAGE, researchers visualize the proteins—separated into distinct bands according to molecular weight—by using a variety of protein stains to make the bands visible. Here we discuss some of the more commonly used stains, including the tried and true as well as recent options for detecting proteins after gel separation.
Coomassie-based stains—the next generation
A traditionally reliable and widely used protein-staining option is Coomassie Blue.
It stains most proteins well and is relatively quick and easy to use. Another advantage of Coomassie Blue stain is that it doesn’t chemically modify proteins in SDS-PAGE, so it is compatible with downstream applications like mass spectrometry and peptide sequencing. A drawback to this stain can be the detection sensitivity, as Coomassie Blue binds to basic and hydrophobic amino acids; as a result, its sensitivity can vary somewhat, depending on the amino acid composition of the proteins being detected.
Recent improvements to Coomassie-based stains have resulted in products that are more sensitive, even easier to work with, and that in some cases use nonhazardous reagents. G-Biosciences offers Coomassie-based RAPIDstain and Labsafe Gel Blue, which both detect down to 4 ng to 8 ng of protein in five to 10 minutes, with the latter stain using environmentally friendly reagents. The company’s Colloidal Blue Stain detects proteins down to 10 ng. “We also offer BlueOUT to remove Coomassie stain from gel spots for InGel digestions,” says Colin Heath, R&D and marketing director at G-Biosciences.
Likewise, Biovision’s Coomassie-based Gel-FAST™ Gel Staining/Destaining Kit offers a quicker protocol than conventional stains. “It is faster [than traditional Coomassie Blue stain] due to the unique formulation, and because we use heat to speed the process,” says Sunetra Ray, technical support scientist at Biovision. Gel-FAST can detect as little as 5 ng of protein, but Ray says that under certain conditions this lower limit can be close to 3 ng.
Bulldog Bio’s AcquaStain also reduces staining to around 10 to 20 minutes, without the need to perform a prewash or destain step. “AcquaStain is designed for research labs or teaching labs that benefit from reducing typical staining protocols from 12 hours to under one hour,” says Bulldog Bio owner Mike Mortillaro.
In Tobin Sosnick’s lab at the University of Chicago, research lab manager Isabelle Gagnon uses AcquaStain to visualize protein bands ranging from 6.5 kDa to 68 kDa with ease. Gagnon likes AcquaStain for several reasons, including its speed and ease of use. “Once the electrophoresis is done, the gel can be soaked right away in the stain,” she says. “There are no longer endless steps to stain and destain the gel—with this stain in one step, the gel is dyed.” She is also pleased that it’s nonhazardous: “It’s eco-friendly, and it doesn’t have a strong, bad smell like the old-fashioned method of staining gels with acetic acid.”
Coomassie-based stains (traditional and newer versions) are also available from other vendors, such as Applied BioProbes, Bio-Rad Laboratories, MilliporeSigma, Protea and Thermo Fisher Scientific.
For a one-step, non-Coomassie-based stain, consider Lonza’s proprietary ProSieve™ EX Safe Stain, which is nontoxic and takes less than 10 minutes. It can even “detect proteins as small as 4 kDa, and as with little as 10 ng of protein,” says Jeffrey Bergeron, product manager in molecular biology at Lonza.
Silver, copper and zinc stains
In terms of colorimetric staining of proteins, silver stains are considered the most sensitive, detecting targets at the sub-nanogram level. With this staining procedure, metallic silver is deposited onto the surface of the gel at specific positions where proteins have migrated. The silver ions interact and bind with specific functional groups on the proteins. This development process is similar to developing traditional photographic film: Silver ions are reduced to metallic silver, which turns the protein bands (but not the surrounding gel) a dark color.
But there are drawbacks to the technique, including a narrower linear dynamic range (and consequently less reproducibility in terms of quantification), and a multistep, time-consuming protocol. And some silver stains use formaldehyde, which makes them incompatible with downstream workflow processes.
G-Biosciences’ FASTsilver™ (with 1-ng sensitivity) and FOCUS FASTsilver™ (with about 0.3-ng sensitivity) stains are compatible with subsequent mass spectrometry when using SilverOUT to remove silver ions.
Although silver stains provide the best sensitivity in many cases, they are not for every protein. Some glycoproteins, for example, can be difficult to stain, so G-Biosciences’ Glycoprotein Staining Kit supports glycoprotein staining using zinc and copper, instead. “The silver stains are notorious for not being able to detect all glycoproteins,” says Heath. “The reversible zinc and copper stains will detect these proteins with similar sensitivity to silver stains.”
Copper and zinc are negative stains, so named because these metal ions precipitate within polyacrylamide gels in which proteins are absent. Detergent molecules on the proteins inhibit the metal ions from precipitating in protein bands. “Because the copper- and zinc-based stains actually stain the gel rather than the proteins,” says Heath, “they are highly compatible with downstream staining.” G-Biosciences also offers Reversible Copper and Zinc Stains (0.1- 0.5 ng sensitivity) for general protein use.
Other companies offering silver, copper and/or zinc staining tools include Bio-Rad Laboratories, MilliporeSigma, Protea and Thermo Fisher Scientific.
Fluorescent and luminescent stains
More recent offerings include fluorescent and luminescent protein stains. These offer sensitivities that rival silver stains—for example, G-Biosciences’ RUBEO fluorescent stain has sensitivity in the 0.1 ng to 0.5 ng range—with the additional advantage of a broader linear dynamic range. These usually involve a simple one-step protocol, but they may take longer than the newer Coomassie-based stains. Most are compatible with downstream mass spectrometry and peptide sequencing, because they don’t chemically react with proteins. Fluorescent stains are often more expensive, though, and they require an appropriate imaging device, such as a fluorescence scanner, to quantify signals.
Specialized fluorescent stains that pinpoint specific protein types are also emerging.
These include Applied BioProbes’ Phospho-TagTM Phosphoprotein Gel Stain, which detects phosphoproteins by attaching to phosphate groups on tyrosine, serine or threonine residues. Fluorescent stains are also offered by other companies, including Bio-Rad Laboratories, Lonza, MilliporeSigma, PromoKine and Thermo Fisher Scientific.
Luminescent stains are another recent option for researchers. Although not as commonly used for protein detection, these stains provide an alternation solution for both detection and sensitivity. A luminescent stain from Biotium, called LumiteinTM, offers sub-nanogram detection sensitivity and requires an imaging device, such as an ultraviolet gel box or a fluorescence scanner.
“Stain-free” staining
Another option for visualizing protein bands in gels is Bio-Rad Laboratories’ stain-free technology, which relies on a small chemical inside the company’s Stain-FreeTM Gels that reacts with tryptophan residues upon exposure to UV light. This reaction results in a red-shifted emission and enhanced protein fluorescence. “It allows you to see what your separation looks like within a few minutes, without the additional steps required in traditional staining,” says Scott Chilton, product manager for protein quantitation at Bio-Rad Laboratories. Users simply place the completed gel in a Bio-Rad Stain-Free enabled imager for activation and quantitation, which takes less than five minutes.
The staining process results in a modification reaction that chemically labels the proteins and enables the researcher to track or follow the protein through additional workflow steps. “If you’re doing a Western blot, you can look at your transfer efficiency without having to do a separate stain on the blot,” Chilton says. “You can look at the blot and gel post-transfer to see how much protein was actually transferred based on the fluorescence intensity.” The method is even compatible with downstream applications such as mass spectrometry and peptide sequencing. According to Chilton, stain-free technology yields a sensitivity and detection range comparable to Coomassie-based stains, and the technology works well with any type of protein that contains tryptophan (present in the vast majority of commonly studied proteins).
Whether you are looking for a stain to confirm the presence of a common protein or for a highly sensitive method of detecting a rare protein, look no further than today’s commercially available protein stains. And check back for future developments on specialized protein stains!
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