Once proteins have been separated and resolved by SDS-PAGE or 2D electrophoresis, the proteins are visualized using different staining procedures. For this purpose, most laboratories worldwide use three common protein staining techniques: Coomassie brilliant blue, silver staining or fluorescent staining. So, how do you know which stain will be best suit for mass spectrometry? Here are some things you need to consider to help you come up with the right decision.
Gel Staining 101: Understanding the Basic Principles
After performing SDS-PAGE, the gel cassette is disassembled and the thin polyacrylamide gel is placed in a tray filled with water or a suitable buffer. The electrophoresed proteins, which exist as concentrated bands embedded within the lanes of the gel matrix, are still bound to anionic SDS detergent. To render these bands visible, the gel matrix needs to be treated with a protein-specific, dye-binding or color-producing chemical such as silver stain or coomassie brilliant blue stain.
For best results, your chosen staining technique should offer a robust, fast, and uncomplicated protocol, and should have high sensitivity and reproducibility, and wide linear dynamic range. In addition, it should be compatible with downstream technologies such as protein extraction and assay, blotting, and/or mass spectrometry.
Which Protein Stain Should You Choose for Mass Spectrometry?
All staining techniques have their own set of advantages and disadvantages so you really need to know more about them so you can make the right choice.
Silver Stain
Silver staining is, by far, the most sensitive colorimetric method that can be used for detecting and visualizing proteins. This technique takes advantage of the fact that silver ions bind strongly with certain protein functional groups such as the carboxylic acid groups (Asp and Glu), imidazole (His), sulfhydryls (Cys), and amines (Lys). With the aid of a developing solution, the silver ions are reduced to silver metal and creates a visible image.
While this technique provides the highest sensitivity (it can be used to detect less than 1ng of protein) and can be extremely useful for applications involving low levels of protein, there are a number of disadvantages associated with its use. This includes the following:
- It is time and labor intensive. The process involves multiple steps and reagents, and the gel needs to be developed after staining. Since the length of time required for developing varies greatly between gels, using this technique does not ensure sufficient reproducibility for quantitative analysis.
- It has a narrow linear dynamic range. This makes silver staining less suitable for quantification.
- It does not stain all proteins. Silver stains are notorious for not being able to stain proteins with common post-translational modifications, such as glycoproteins and phosphoproteins.
- It offers limited compatibility with downstream applications. Traditional silver staining requires the use of glutaraldehyde or formaldehyde which may cause chemical crosslinking of the proteins in the gel matrix. This limits the number of compatible methods for analysis by mass spectrometry (MS). Newer mass spec compatible silver stains are now commercially available that offer greater compatibility, but still have the same disadvantages as traditional silver stains.
Coomassie Stains
This is perhaps the most widely known protein staining technique being used in laboratories around the world. There are two main types of Coomassie stains - the original Coomassie stain and the colloidal Coomassie stains.
In the classical Coomassie staining technique, the protein gels are incubated with a Coomassie staining solution. Since this process stains the whole gel, it is then washed with methanol/acetic acid destaining solution to allow for the visualization of the protein bands.
While this technique is considerably less sensitive as compared to silver staining (it has a detection limit of about 100ng) and has low reproducibility, many researchers used to use this technique since it is cheap and simple to perform. In addition, it does not modify the target proteins and is compatible with mass spectrometry. This technique also proves to be quite sufficient for simple tasks such as visualization of recombinant proteins or generated antibodies.
To overcome the limitations of silver staining and the original Coomassie staining technique, researchers now prefer to use colloidal Coomassie stains instead. This technique offers higher sensitivity (detection limit of about 4ng) and reproducibility (the colloidal dye does not penetrate the gel so it does not require destaining) as compared to the original coomassie staining technique. In addition, it is also ideal for applications involving low protein levels and is very much compatible with mass spectrometry.
Coomassie stains major disadvantages are the lower sensitivity and the stain has to be removed before mass spec analysis.
There are a large number of fluorescent stains available on the market and the majority of them are compatible with mass spectrometry. The basic principle is the binding of a fluorescent dye to the proteins that is activated by a specific wavelength to emit fluorescent light. The fluorescent protein stains have similar sensitivity to silver stains, have been shown to produce improved mass spectra.
Fluorescent stains do have disadvantages including the length of the protocols (up to 5 hours in some cases), cost and they still require destaining before mass spec analysis.
Reversible Stains
Reversible stain are often overlooked for staining protein gels for mass spectrometry, but they do offer several advantages over the above stains.
The most common reversible stain is zinc stain, but other metal stains, such as copper stain, are available. The stains basically work by depositing metal precipitate into the protein gel, however the SDS prevents the precipitate from binding to the proteins. This generates a reverse or negative gel image as the gel is stained white (with zinc) or green (with copper) and the protein bands are clear, unstained. This offers a huge advantage as no destaining is required and the proteins have not been modified by a dye binding to it.
A second advantage is that these stains offer comparable sensitivity to the silver and fluorescent stains and is not affected by the post translational modifications of the proteins. This means all proteins can be detected.
The reversible stains are often overlooked as they do require a little extra skill to use, but the extra effort is often worth the clean, unstained, unmodified protein for mass spec analysis.
