Protein Gel Stains
General principles of gel staining
The first step after performing denaturing polyacrylamide gel electrophoresis (SDS-PAGE) is to disassemble the gel cassette and place the thin (1 mm thick) polyacrylamide gel in a tray filled with water or buffer. The electrophoresed proteins exist as concentrated "bands" embedded within each lane of the porous polyacrylamide gel matrix. Typically, the proteins are still bound to anionic SDS detergent, and the entire gel matrix is saturated in a particular buffer.
To make the proteins visible, a protein-specific, dye-binding or color-producing chemical reaction must be performed on the proteins within the gel. Depending on the particular chemistry of the stain, various steps are necessary to hold the proteins in the 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.
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 stain reagent to allow the dye or chemical to diffuse into the gel and bind (or react with) the proteins
- Destaining to remove excess dye from the background gel matrix
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 stain-reagent step and a developer step to produce the colored reaction product.
The Thermo Scientific Pierce Electrophoresis Tech Handbook provides information to improve the speed, convenience and sensitivity of your protein gel electrophoresis and staining applications. The Handbook covers all aspects of electrophoresis from sample and gel preparation to choice of molecular weight markers. In addition, it contains an extensive section on protein gel staining techniques and products.
The most common method for in-gel protein detection is staining with coomassie dye. Several coomassie stain reagent recipes exist in the literature and use either the G-250 (“colloidal”) or R-250 form of the dye. Colloidal coomassie can be formulated to effectively stain proteins within one hour and require only water (no methanol or acetic acid) for destaining.
In acidic buffer conditions, coomassie dye binds to basic and hydrophobic residues of proteins, changing from dull reddish-brown to intense blue (see previous images on this page). As with all staining methods, coomassie dye reagents detect some proteins better than others based on their chemistry of action and differences in protein composition. Thus, coomassie dye reagents can detect as few as 8-10 nanograms per band for some proteins and 25 nanograms 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 stain reagent is added, usually for about 1 hour; finally, a water or simple methanol:acetic acid destaining step is used to wash away excess non-bound 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.
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 location of protein bands. Silver ions (from silver nitrate in the stain reagent) interact and bind with certain protein functional groups. 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 brown-black color.
Silver staining protocols require several steps that are affected by reagent quality as well a 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 minimize the effects of minor differences in day-to-day use. Kits with optimized protocols are robust and easy to use, detecting less than 0.5 nanograms 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 compatible with destaining and elution methods for analysis by mass spectrometry (MS). Therefore, optimization of sensitivity vs. protein-recoverability is critical when 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.
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 protein occur. 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 the gel over a dark background. Zinc staining is as sensitive as typical silver stains (detects less than 1 ng of protein) and there are no fixation steps. Furthermore, the stain is easily removed, making this method compatible with mass spectrometry or Western blotting.
The recent improvements in fluorescence imagers and fluorescent applications have resulted in greater demand for fluorescent stains beyond the traditional ethidium bromide stain for nucleic acids. A number of total protein fluorescent stains have been introduced in recent years. Newer fluorescent total protein stains provide exceptional fluorescent staining performance with a 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 imagers.
Most fluorescent stains involve simply 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 mass spectrometry. Accordingly, they are frequently used in both 1D and 2D applications.
Sometimes it is desirable to detect a subset of proteins rather than all of the proteins in a gel. Glycoproteins and phosphoproteins are categories of proteins that are classified on the basis of a particular type of chemical moiety (i.e., polysaccarides and phosphate groups, respectively). 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 which 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 and glycoprotein stain kits are available commercially.
Various protein gel staining methods, both colorimetric and fluorescent have also been developed to detect phosphorylated proteins and his-tagged fusion protein.
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