Protein Staining

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Overview

Proteins separated by gel electrophoresis can be visualized using different staining procedures. The choice of staining technique depends on the availability of imaging equipment in the lab in many cases. This section provides general considerations for staining and describes different total protein stains such as Coomassie, fluorescent, silver, and negative stains, and provides troubleshooting tips and protocols for different staining procedures. It also discusses the features and benefits of stain-free technology for protein visualization.

Related Topics: SDS-PAGE Analysis.

General Considerations

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

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.

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.

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.

Stain-Free Technology

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 Mini PROTEAN® TGX Stain-Free™ Gels 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 enabled imager, such as ChemiDoc™ MP, ChemiDoc™ Touch, ChemiDoc™ XRS+, or Gel Doc™ EZ Imagers) 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.

ChemiDoc Touch Stain-Free Enabled Imaging System

Bio-Rad's ChemiDoc Touch Stain-Free Enabled Imaging System. Left: the ChemiDoc Touch System comprises the imager with UV/stain-free tray and Image Lab Software. Right: Protein gel imaged using stain-free technology.

Specific Protein Stains

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

Flamingo Stains Oriole Stains Coomassie Blue Stains Silver Stains SYPRO Stains Stain-free Stains

Total Protein Stain Sensitivity
(Lower Limit)
Time Comments Imaging
Stain-Free
  Similar to Coomassie and dependent upon tryptophan No staining or N/A Fast and reproducible; visualize proteins in 5 minutes or less with a stain-free enabled imager; no staining required  
Coomassie Stains Densitometer
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  
QC Colloidal Coomassie 3 ng 1–20 hr Colloidal endpoint stain; premixed, nonhazardous formulation — no methanol required  
Silver Stains Densitometer
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; compatible only 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.

Troubleshooting

Problem Cause Solution
Bands not visible No protein in gel Stain with another method to confirm there is protein
Imaging system malfunctioning Check instrument manual for troubleshooting, or contact imaging instrument manufacturer
Incorrect imaging parameters were used Check instrument manual
Poor staining sensitivity Dirty staining trays Clean staining trays and other equipment with laboratory glassware cleaner
Insufficient stain volume Follow recommendations for stain volume (appropriate to gel size)
Insufficient staining time Increase staining time
Reuse of staining solution Repeat staining protocol with fresh staining solution
High or uneven background staining Dirty equipment or staining trays Clean staining trays and other staining equipment with laboratory glassware cleaner
Too much time in staining solution Restrict duration of incubation in staining solutions as recommended in protocol

Wash gel in water or respective destaining solution for ≥30 min
Reagent impurities Use high-purity water and reagents for staining
Speckles or blotches in gel images Particulate material from reagents, staining tray, dust or gloves Clean staining trays thoroughly

Limit time that gels and staining solution are exposed to open air

Use dust-free gloves and handle gels only by edges
Uneven staining Insufficient shaking during staining Agitate gel during staining
Gel shrinkage   Transfer gel to water for rehydration

References

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.

Gottlieb M and Chavko M (1987). Silver staining of native and denatured eukaryotic DNA in agarose gels. Anal Biochem 165(1), 33–37.

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 shows 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, 1128–1144.

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, 3666–3672.

Related Content

Literature
Number Description Download
6517 ChemiDocâ„¢ Touch Imaging System Brochure, Rev A Click to download
6001 Rapid Validation of Purified Proteins using Criterion Stain Free Gels, Rev A Click to download
5705 Sensitivity and Protein-to-Protein Consistency of Flamingo Fluorescent Gel Stain Compared to Other Fluorescent Stains, Rev A Click to download
5754 Comparison of SYPRO Ruby and Flamingo Fluorescent Gel Stains With Respect to Compatibility With Mass Spectrometry, Rev A Click to download
5921 Oriole Fluorescent Gel Stain: Characterization and Comparison with SYPRO Ruby Gel Stain, Rev A. Click to download
2423 Bio-Safe Coomassie Stain Brochure, Rev D Click to download
5346 Flamingo Fluorescent Gel Stain Product Information Sheet, Rev A Click to download
6209 Total Protein Staining Click to download
6217 Total Protein Detection Click to download
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