Azure Biosystems
Electrophoresis is used daily in many labs to separate protein or nucleotide samples by size for downstream analysis. A variety of methods are used to detect the separated biomolecules, including radiolabeling and fluorescent dyes. The most straightforward means of detection is done by first staining the protein or DNA with a colorimetric dye and then using a gel documentation system, also called a gel doc, to image the gel. Visible gel imaging is also known as true color imaging.
Because both agarose and polyacrylamide gels are transparent, the gels stained with colorimetric dyes can be easily imaged under ambient light or using a white-light transilluminator (Figure 1).
For protein gels, visible detection is rapid and sensitive. Unlike fluorescent stains, colorimetric stains do not require special imaging equipment and colorimetrically stained gels can be examined by eye or documented using any available camera by placing the gel over a white surface or a transilluminating light box. For more accurate quantitation, colorimetrically stained gels can also be imaged using densitometry.
| Benefits of using visible gel imaging for protein gels with colorimetric stains |
|---|
| Visible detection is rapid and sensitive. |
| Colorimetric stains do not require special imaging equipment. |
Table 1. Advantages of imaging protein gels with visible gel imaging
For DNA gels, colorimetric stains allow the scientist to avoid the use of UV light when imaging their samples. DNA is most frequently imaged using ethidium bromide or other intercalating dyes that fluoresce under UV light. UV light can nick and damage DNA.
For experiments that involve staining a DNA gel to identify a specific DNA band, cutting that band out of the gel, and extracting the DNA from the gel for downstream experiments such as subcloning, any DNA damage can reduce the efficiency of the downstream steps. Therefore, a non-damaging dye that avoids the use of UV light can increase the probability of success; however, visible-dye-based DNA stains are typically less sensitive than fluorescent stains.
Coomassie brilliant blue is a commonly used colorimetric stain for protein gels (Figure 2). The stain binds proteins non-covalently so is compatible with downstream analyses such as mass spectrometry which is frequently used to identify the protein in a spot or band from an gel. Multiple formulations of Coomassie stain are commercially available and a variety of staining techniques have been published. Generally, the gel is incubated in a stain solution and then is destained in a destaining solution to reduce background staining. The entire protocol can take from about 30 minutes to overnight, depending on the approach.
Silver staining is the most sensitive colorimetric stain for protein gels. It’s able to detect low nanogram amounts of protein. A variety of protocols are available, taking from about 1 hour to overnight. Most silver stains are not compatible with mass spectrometry, but variations of the approach have been developed that, though less sensitive than traditional silver staining, can be used with mass spec.
DNA is most frequently visualized using fluorescent dyes but several colorimetric dyes can be used to detect DNA bands.
Methylene blue can be used to stain DNA gels after electrophoresis. The staining protocol takes from an hour to overnight, and the amount of destaining required depends on the method used. Methylene blue staining is reversible, and less sensitive than ethidium bromide staining.
In addition to the stains mentioned, several proprietary colorimetric stains are also commercially available.
A key application of visible gel imaging is densitometry. Densitometry is used to quantitate the amount of sample in a band or spot on a stained gel. This type of analysis requires a digital image and involves comparing the pixel depth of the unknown band to the pixel depth of standard bands containing known concentrations.
For true quantitative results, the stained gel must be imaged using the new chemiSOLO, Azure Sapphire FL, or other digital imaging system. The imaging can be direct, where light is sent straight through the gel towards a detector, or reflected, using a densitometry plate. The optical density of the resulting gel image depends on how much light is blocked by the stained sample.
Gel docs and imaging systems, like the Azure Imaging Systems, come in a variety of configurations. These modern systems visualize nucleic acid and protein samples in gels by using visible wave lengths, most of the time using CCD or CMOS cameras.
Which Azure Imager is right for you? Click here to find out!
If you’re imaging chemiluminescent Western blots and tight on budget and space, your go-to choice would be the new chemiSOLO. The chemiSOLO is a new personal Western blot imager able to detect low-expressing proteins with femtogram sensitivity and captures marker images at the push of a button. Learn more about the chemiSOLO and how it can easily add high-resolution images to your studies by clicking below.
In chemiluminescent detection, the primary antibody binds to the target protein on the membrane, and the location of the primary antibody is detected using a secondary antibody conjugated to an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP).
Once a substrate for the enzyme is added, the enzyme acts on the substrate, causing light to be emitted. This light is then detected using a gel imager through its CCD camera. Read more
In a Coomassie stained gel, the protein bands appear blue in a clear background may be examined by eye on the bench or on a light box. The gel may be imaged photographed with white light. Check out the difference between Coomassie-stained gels imaged with white light and NIR fluorescence
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What is Coomassie? Coomassie is a blue stain used in proteomics-related studies to detect proteins during electrophoresis, SDS-PAGE, and Bradford assays. In Western blot analysis, Coomassie
Western blotting is a tried and true way to detect and evaluate protein expression and is widely used by researchers. While it has been around for
Chemiluminescent detection depends on an enzymatic reaction so timing and the amount of both enzyme and substrate used have important effects on data quality. Light
