Visible Gel Imaging

What is visible gel imaging?

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).

Coomassie-stained gel from Azure chemiSOLO
Figure 1. A protein SDS-PAGE gel stained with Coomassie dye and imaged using the Visible Color Imaging option on the chemiSOLO.

Advantages to visible gel imaging

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.

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.

What stains are used for visible imaging of protein gels?

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.

Coomassie-stained gel imaged on the Azure chemiSOLO
Figure 2. Coomassie-stained gel imaged on the Azure chemiSOLO using white light

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 stains used for visible imaging of DNA gels

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.

Scientist using pliers to place gel inside Azure chemiSOLO
The new Azure chemiSOLO detects low-expressing proteins with femtogram sensitivity and captures marker images at the push of a button. In addition to Western blots, the chemiSOLO captures pictures of colorimetric blots or visible stained protein gels, like Coomassie blue or silver stain.

Quantitation of gels stained with colorimetric stains

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, 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.

What can I use to image gels?

Gel docs and imaging systems, like the Azure Imaging Systems, come in a variety of configurations. Gel docs visualize nucleic acid and protein samples in gels by using visible wave lengths.

If you’re imaging chemiluminescent Western blots and tight on budget and space, your go-to personal Western blot imager would be the new Azure chemiSOLO. The chemiSOLO is able to detect low-expressing proteins with femtogram sensitivity and captures marker images at the push of a button.

Which gel doc is right for you? Click here to find out!

Frequently Asked Questions

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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