A protein staining technique should offer the following:
- High sensitivity and reproducibility
- Wide linear dynamic range
- Compatibility with downstream technologies such as protein extraction and assay, blotting, or mass spectrometry
- Robust, fast, and uncomplicated protocol
Staining protocols usually involve the following three steps:
- Protein fixation, usually in acidic methanol or ethanol (a few staining protocols already contain acid or alcohols for protein fixation and do not require this separate step)
- Exposure to dye solution
- Washing to remove excess dye (destaining)
See the protocols tab below for individual protocols.
Total protein stains allow visualization of the protein pattern in the gel. The table below compares the advantages and disadvantages of several total protein staining techniques.
Coomassie Stains
The most popular anionic protein dye, Coomassie Brilliant Blue, stains almost all proteins with good quantitative linearity at medium sensitivity. There are two variants of Coomassie Brilliant Blue: R-250 (R for reddish), which offers shorter staining times, and G-250 (G for greenish), which is available in more sensitive and environmentally friendly formulations (Simpson 2010, Neuhoff et al. 1988). Coomassie dyes are also the favorite stains for mass spectrometry and protein identification. Bio-Safe™ Coomassie stain is a nonhazardous formulation of Coomassie Blue G-250 that requires only water for rinsing and destaining. It offers a sensitivity that is better than conventional Coomassie R-250 formulations and equivalent to Coomassie Blue G-250, but with a simpler and quicker staining protocol.
Fluorescent Stains
These stains fulfill almost all the requirements for an ideal protein stain by offering high sensitivity, a wide linear dynamic range over four orders of magnitude, a simple and robust protocol, and compatibility with mass spectrometry. In comparison to Coomassie or silver staining techniques, however, fluorescent dyes are more expensive and require either a CCD (charge coupled device) camera or fluorescence scanner for gel imaging. For these reasons, fluorescent stains are often used in proteomics applications and on 2-D gels, where the relative quantitation of proteins in complex mixtures over several orders of abundance is performed, and protein identity is determined using in-gel proteolytic digestion and mass spectrometry. Examples include Flamingo™ and Oriole™ fluorescent gel stains.
Silver Stains
Silver stains offer the highest sensitivity, but with a low linear dynamic range (Merril et al. 1981. Rabilloud et al. 1994). Often, these protocols are time-consuming, complex, and do not offer sufficient reproducibility for quantitative analysis. In addition, their compatibility with mass spectrometry for protein identification purposes is lower compared to Coomassie stains and fluorescent dyes (Yan et al. 2000). Silver stain offers sensitivity that exceeds that of Coomassie and is equivalent to most fluorescent stains.
The ability to visualize gels without a staining step has gained popularity recently. Stain-free technology has several advantages over staining procedures. In particular, it eliminates the need for a lengthy destaining step, makes visualization faster and more efficient, and eliminates disposal of organic waste generated during destaining. The stain-free system usually comes with an automated imaging and analysis system (see below), and gives less background compared to traditional Coomassie staining procedures, thereby reducing variability between and within gels.
A special trihalo compound present in stain-free gels, such as Bio-Rad's Criterion Stain-Free™ Tris-HCl and Criterion™ TGX Stain-Free™ gels, covalently binds to proteins' tryptophan residues when activated with UV light. Activation of the trihalo compounds in the gels adds 58 Da moieties to available tryptophan residues and is required for protein visualization. Proteins that do not contain tryptophan residues are not detected. This allows protein detection (with a stain-free compatible imager, such as Gel Doc™ EZ imager or ChemiDoc™ MP Imager) in a gel both before and after transfer, as well as total protein detection on a blot using wet PVDF membranes.
The sensitivity of the stain-free system is comparable to staining with Coomassie Brilliant Blue for proteins with a tryptophan content >1.5%; sensitivity superior to Coomassie staining is possible for proteins with a tryptophan content >3%. Stain-free technology is compatible with downstream immunodetection, though some antibodies may show a slightly lower affinity for the haloalkane-modified proteins.
Bio-Rad's stain-free system. Left: the system comprises the Gel Doc™ EZ imager with stain-free tray, Image Lab™ software, and precast gels (Criterion format). Right: Gel (top) and blot (bottom) imaged using stain-free technology.
Specific protein stains are used to visualize specific protein classes such as glycoproteins (Hart et al. 2003) and phosphoproteins (Steinberg et al. 2003, Agrawal and Thelen 2009), which are of special interest to researchers working in the life sciences (examples include Pro-Q Diamond and Pro-Q Emerald).
Bio-Rad Gel Stain Selection Guide
| Total Protein Stain |
Sensitivity
(Lower Limit) |
Time |
Comments |
Imaging |
| Coomassie Stains |
Densitometry |
| Bio-Safe Coomassie G-250 |
8–28 ng |
1–2.5 hr |
Nonhazardous staining in aqueous solution; premixed, mass spectrometry compatible |
|
| Coomassie Brilliant Blue R-250 |
36–47 ng |
2.5 hr |
Simple and consistent; mass spectrometry compatible; requires destaining with methanol |
|
| Silver Stains |
Densitometry |
| Silver Stain Plus™ kit |
0.6–1.2 ng |
1.5 hr |
Simple, robust; mass spectrometry compatible (Gottlieb and Chavko 1987) |
|
| Fluorescent Stains |
CCD and Laser-Based Scanners |
| Oriole fluorescent gel stain* |
0.5–1 ng |
1.5 hr |
Rapid protocol, requires no destaining, mass spectrometry compatible; only compatible with UV excitation |
|
| Flamingo fluorescent gel stain |
0.25–0.5 ng |
5 hr |
High sensitivity; broad dynamic range; simple protocol requires no destaining; mass spectrometry compatible; excellent for laser-based scanners |
|
| SYPRO Ruby protein gel stain |
1–10 ng |
3 hr |
Fluorescent protein stain; simple, robust protocol; broad dynamic range; mass spectrometry compatible |
|
|
* Do not use Oriole gel stain with native gels.
|
Staining Equipment
Commercially available stainers, such as Dodeca™ high-throughput stainers can be used in staining procedures to increase consistency in staining and to avoid breakage of gels from excess handling.
Dodeca high-throughput stainers.
Agrawal GK and Thelen JJ (2009). A high-resolution two dimensional Gel- and Pro-Q DPS-based proteomics workflow for phosphoprotein identification and quantitative profiling. Methods Mol Biol 527, 3–19.
Hart C et al. (2003). Detection of glycoproteins in polyacrylamide gels and on electroblots using Pro-Q Emerald 488 dye, a fluorescent periodate Schiff-base stain. Electrophoresis 24, 588-598.
Merril CR (1987). Detection of proteins separated by electrophoresis. Adv. Electrophoresis 1, 111–139.
Merril CR et al. (1981). Ultrasensitive stain for proteins in polyacrylamide gels show regional variation in cerebrospinal fluid proteins. Science 211, 1437–1438.
Miller I et al. (2006). Protein stains for proteomic applications: which, when, why? Proteomics, 6, 5385–5408.
Neuhoff V et al. (1988). Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear backgrounds at nanogram sensitivity using G-250 and R–250. Electrophoresis 9, 255–262.
Rabilloud T et al. (1994). Silver-staining of proteins in polyacrylamide gels: a general overview. Cell Mol Biol 40, 57–75.
Simpson RJ (2010). Rapid coomassie blue staining of protein gels. Cold Spring Harb Protoc, pdb prot5413.
Steinberg TH et al. (2003). Global quantitative phosphoprotein analysis using multiplexed proteomics technology. Proteomics 3, 1128–44.
Yan JX et al. (2000). A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry. Electrophoresis 21, 3666–72.
