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.

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

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

Coomassie stain from chemiSOLO
Figure 1. The image above shows 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. 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, detecting 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.

What stains are 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.

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