Once proteins have been separated by electrophoresis, they can be visualized using different methods of in-gel detection, each with advantages and disadvantages. Over the past several decades, demand for improved sensitivity for small sample sizes and compatibility with downstream applications and detection instrumentation have driven the development of several basic staining methods. Here we discuss the general principles of protein gel staining and describe several staining methods.
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Page contents
- General principles of gel staining
- Coomassie dye staining
- Silver staining
- Fluorescent dye staining
- Zinc staining
- Functional group–specific stains
General principles of gel staining
To make proteins visible, a protein-specific, dye-binding or color-producing chemical reaction can be performed on the proteins within the gel. Depending on the particular chemistry of the stain, various steps are necessary to retain, or fix, the proteins in the gel matrix and to facilitate the necessary chemical reaction. All steps are done in solution, i.e., with the gel suspended in a tray filled with one liquid reagent or another. Typically, the proteins are still bound to the anionic detergent (SDS), and the entire gel matrix is saturated in running buffer after electrophoresis.
Given the common constraints of this format, most staining methods involve some version of the same general incubation steps:
- A water wash to remove electrophoresis buffers from the gel matrix
- An acid or alcohol wash to condition or fix the gel to limit diffusion of protein bands from the matrix
- Treatment with the staining reagent to allow the dye or chemical to diffuse into the gel and bind to (or react with) the proteins
- Destaining to remove excess dye from the gel matrix background
Depending on the particular staining method, two or more of these functions can be accomplished with one step. For example, a dye reagent that is formulated in an acidic buffer can effectively fix and stain in one step. Conversely, certain functions require several steps. For example, silver staining requires both a staining reagent step and a developer step to produce the colored reaction product.
Coomassie dye stains
The most common method of in-gel protein detection is staining with Coomassie dye. These stains either use the G-250 (“colloidal”) or the R-250 form of the dye. Colloidal Coomassie stains can be formulated to effectively stain proteins within 1 hour and requires only water (no methanol or acetic acid) for destaining.
Example gel stained with Simply Blue Safe Coomassie Stain. Samples were separated on a NuPAGE 4-12% Bis-Tris gel and stained with SimplyBlue SafeStain.
- Lane 1: 6 µg protein mix
- Lane 2: 1 µg rabbit IgG
- Lane 3: 1 µg reduced BSA
- Lane 4: 5 µg E. coli lysate
- Lane 5: 20 ng reduced BSA
- Lane 6: 10 ng reduced BSA
- Lane 7: 7 ng reduced BSA
- Lane 8: 3 ng reduced BSA
- Lane 9, 10: Mark12 Unstained Standard
| Sensitivity | Typical protocol time | Detection | Compatibility with downstream applications | Advantages | |
|---|---|---|---|---|---|
| Coomassie staining | 5-25 ng | 10-135 min | Visual | Mass spectrometry (MS) and sequencing compatible, western blotting (only non-fixative methods) |
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In acidic conditions, Coomassie dye binds to basic and hydrophobic residues of proteins, changing in color from a dull reddish-brown to intense blue. As with all staining methods, Coomassie staining detects some proteins better than others, based on the chemistry of action and differences in protein composition. Thus, Coomassie staining can detect as little as 8–10 ng per band for some proteins and 25 ng per band for most proteins.
Coomassie dye staining is especially convenient because it involves a single ready-to-use reagent and does not permanently chemically modify the target proteins. An initial water wash step is necessary to remove residual SDS, which interferes with dye binding. Then, the staining reagent is added, usually for about 1 hour; finally, a water or simple methanol: acetic acid destaining step is used to wash away excess unbound dye from the gel matrix. Because no chemical modification occurs, excised protein bands can be completely destained and the proteins recovered for analysis by mass spectrometry or sequencing.
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Silver staining
Silver staining is the most sensitive colorimetric method for detecting total protein. The technique involves the deposition of metallic silver onto the surface of a gel at the locations of protein bands. Silver ions (from silver nitrate in the staining reagent) interact and bind with certain protein functional groups. The strongest interactions occur with carboxylic acid groups (Asp and Glu), imidazole (His), sulfhydryls (Cys), and amines (Lys). Various sensitizer and enhancer reagents are essential for controlling the specificity and efficiency of silver ion binding to proteins and effective conversion (development) of the bound silver to metallic silver. The development process is essentially the same as for photographic film: silver ions are reduced to metallic silver, resulting in a brown-black color.
Example gel stained with SilverXpress Silver Staining Kit. Samples were separated on an NuPAGE 4–12% Bis-Tris Protein Gel and stained with the SilverXpress Kit.
- Lanes 1–5: Invitrogen Mark12 Unstained Standard (blend of 12 purified proteins), serial 2-fold dilutions ranging from 1:4 to 1:64
- Lane 6: 1.6 ng BSA
- Lane 7: 0.8 ng BSA
- Lane 8:E. coli lysate diluted 1:20
- Lane 9:E. coli lysate diluted 1:80
- Lane 10: replicate of Lane 1
| Sensitivity | Typical protocol time | Detection | Compatibility with downstream applications | Advantages | |
|---|---|---|---|---|---|
| Silver staining | 0.25-0.5 ng | 30-120 min | Visual | Certain formulations are MS compatible | Lowest detection limits not requiring specialized equipment |
Silver staining protocols require several steps, which are affected by reagent quality as well as incubation times and thickness of the gel. An advantage of commercially available silver staining kits is that the formulations and protocols are optimized and consistently manufactured, helping to maximize consistency of results from experiment to experiment. Kits with optimized protocols are robust and easy to use, detecting less than 0.5 ng of protein in typical gels.
Silver stains use either glutaraldehyde or formaldehyde as the enhancer. These reagents can cause chemical crosslinking of the proteins in the gel matrix, limiting compatibility with destaining and elution methods for analysis by mass spectrometry (MS). Therefore, optimization of sensitivity vs. protein recoverability is critical when employing silver staining as part of an MS workflow.
Silver stain formulations can be made such that protein bands stain black, blue-brown, red, or yellow, depending on their charge and other characteristics. This is particularly useful for differentiating overlapping spots on 2D gels.
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Fluorescent dye staining
Recent improvements in fluorescence imaging instruments and fluorescent applications have resulted in greater demand for fluorescent stains. Several fluorescent stains for total protein have been introduced in recent years. Newer fluorescent total-protein stains provide exceptional fluorescent staining performance with fast and easy procedures. The most useful are those whose excitation and emission maxima corresponding to common filter sets and laser settings of popular fluorescence imaging instruments.
Example gel stained with SYPRO Orange Protein Stain and visualized using a blue led transilluminator.
| Sensitivity | Typical protocol time | Detection | Compatibility with downstream applications | Advantages | |
|---|---|---|---|---|---|
| Fluorescent dye stains | 0.25-0.5 ng | 60 min | UV or blue/green-light transilluminators or imaging instruments with appropriate filters | Most stains are MS compatible, western blotting | Broad linear dynamic range with low detection limits |
Most fluorescent stains involve simple dye-binding mechanisms rather than chemical reactions that alter protein functional groups. Therefore, most are compatible with destaining and protein recovery methods for downstream analysis by MS or western blotting. Accordingly, these stains are frequently used in both 1D and 2D applications.
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Explore: Fluorescent labeling for protein normalization
Zinc staining
Zinc staining is unlike all other staining methods. Instead of staining the proteins, this procedure stains all areas of the polyacrylamide gel in which there are no proteins. Zinc ions complex with imidazole, which precipitates in the gel matrix except where SDS-saturated proteins are located. The milky-white precipitate renders the background opaque while the protein bands remain clear. The process is short (about 15 minutes), and the gel can be photographed by viewing it over a dark background. Zinc staining is as sensitive as typical silver staining (detects less than 1 ng of protein), and no fixation steps are required. Furthermore, the stain is easily removed, making this method compatible with MS or western blotting.
Example gel stained with a zinc stain. A 2-fold dilution series of a protein mixture was separated by protein gel electrophoresis using a 15-well mini gel. Subsequently the gel was stained using the Thermo Scientific Pierce Zinc Reversible Stain Kit, and then photographed with the gel placed over a dark blue background. The sensitivity on this gel is 0.25 ng, as indicated by the bands that are visible in the last lane.
| Sensitivity | Typical protocol time | Detection | Compatibility with downstream applications | Advantages | |
|---|---|---|---|---|---|
| Zinc stains | 0.25-0.5 ng | 15 min | Visual | MS compatible, western blotting | No chemical modification of proteins; bands are visible because the background is stained (not the bands) |
Functional group–specific stains
Sometimes it is desirable to detect a subset of proteins rather than all of the proteins in a sample. Differential staining methods for specific protein modifications such as glycoproteins and phosphoproteins are available. Various protein gel staining methods, both colorimetric and fluorescent, have also been developed to detect His-tagged fusion proteins and Lumino tagged fusion proteins.
When a dye-binding or color-producing chemistry can be designed to detect one of these functional groups, it can be used as the basis for a specific gel stain.
Proteins that have been post-translationally modified by glycosylation can be detected by a procedure that involves chemical activation of the carbohydrate into a reactive group. The method works by fixing the proteins in the gel and then oxidizing the sugar residues with sodium meta-periodate. The resulting aldehyde groups can then be reacted with an amine-containing dye. In older literature, this method is known as the periodate acid–Schiff (PAS) technique. A subsequent reduction step stabilizes the dye–protein bond. Both colorimetric and fluorescent dyes have been used for this technique, and glycoprotein stain kits are available commercially.
Example gel stained to visualize Phosphoprotein and total proteins in a 2D gel. Protein lysates obtained from a Jurkat T-cell lymphoma line were separated by 2D gel electrophoresis and subsequently stained with Invitrogen Pro-Q Diamond phosphoprotein gel stain (blue) followed by SYPRO Ruby protein gel stain (red). The gel was dried and imaged on an FLA-3000 scanner (Fuji). Shown is a digitally pseudocolored composite overlaid image.
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Recommended reading
- Diezel W, Kopperschläger G, Hofmann E. An improved procedure for protein staining in polyacrylamide gels with a new type of Coomassie Brilliant Blue. Anal. Biochem. 48(2), 617–620 (1972).
- O'Farrell PH. High resolution 2D electrophoresis of proteins. J. Biol. Chem. 250(10), 4007–4021 (1975).
- Chevalier F, Rofidal V, Vanova P, Bergoin A, Rossignol M. Proteomic capacity of recent fluorescent dyes for protein staining. Phytochemistry 65(11), 1499–1506 (2004).
- Rabilloud T. A comparison between low background silver diammine and silver nitrate protein stains. Electrophoresis 13(7), 429–439 (1992).
- Fernandez-Patron C, Castellanos-Serra L, Hardy E et al.Understanding the mechanism of the zinc-ion stains of biomacromolecules in electrophoresis gels: generalization of the reverse-staining technique. Electrophoresis 19(14), 2398–2406 (1998).
Additional resources
- Gel imaging and documentation
- Total protein normalization for western blotting
- Handbook: Protein Gel Electrophoresis Technical Handbook
- Handbook: Protein Transfer Technical Handbook
- Handbook: Protein Detection Technical Handbook
- Handbook: Protein Research
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