Gel Electrophoresis Stain: Overview & Application
Last updated on March 8, 2023 by
Gel Electrophoresis Stain: Overview
Gel electrophoresis is a commonly used technique in molecular biology and life sciences labs to separate proteins and nucleic acids (both DNA and RNA) based on their molecular size. The gel acts like a sieve for the molecules that travel through it.
The speed of molecules is inversely proportional to their size. Meaning, the smaller molecules will travel far in the gel across to the other end, whereas the bigger ones remain closer to gel wells. The molecules in the gel are separated in the form of bands that are visualized using gel stains.
The electrophoresis gel stains are reagents used in lab assays to visualize the separated protein bands or nucleic acids in gels. They bind to the molecules and create a contrasting background, which helps in their visualization.
Based on the molecule being visualized, the gel stains are classified as nucleic acid gel stains and protein gel stains. The nucleic acid stain is further divided into DNA gel stain and RNA gel stain. The classification is necessary to understand which reagent will be suitable for the macromolecule in the study.
Transilluminators are used to excite dye molecules once they bind with the target molecules. It helps in the visualization, analysis, and quantification of the molecules of interest.
In this article, we will cover more on the types of gel electrophoresis stains, including their types, applications, and industries frequently using these chemicals in their research or investigation processes.
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What are the Types of Gel Electrophoresis Stains?
Many types of gel electrophoresis stains are available today in the market. However, based on the molecule they are used to visualize, they are categorized into two groups: nucleic acid gel stains and protein gel stains.
Nucleic Acid Gel Stains
Nucleic acid stains are chemicals that insert themselves into the structure of the nucleic acids and fluoresce with UV light excitation. They are involved in the assay either during:
- Pre-loading, which occurs before running a gel by adding the stain to the loading dye and then mixing it up with DNA,
- Pre-casting, which occurs while running the gel by adding the stain to the gel so the DNA fragments pick it by themselves when they start migrating, or
- Post-staining, which occurs after the agarose gel electrophoresis is over by soaking the gel in a staining solution.
Ethidium bromide (EtBr) is a commonly used DNA stain, which is used while pre-casting or post-staining. However, it should be carefully used as it is also a potent mutagen. It’s especially a problem when you need to use DNA for downstream applications, such as cloning.
A safer and more sensitive alternative to ethidium bromide (EtBr) is the fluorescent stain of the SYBR family. For example, SYBR Gold has higher sensitivity for ssDNA, dsDNA, and RNA detection. It’s excellent for downstream applications, such as denaturing gradient gel electrophoresis and routine gel analysis.
Similarly, SYBR Green I can detect DNA in both agarose and polyacrylamide gels with exceptional sensitivity, and SYBR safe has sensitivity comparable to EtBr, but it’s much safer to use in lab assays. Moreover, as the dye uses blue light rather than UV light, it protects DNA from any further damage.
One limitation of these stains is that they inhibit polymerase chain reaction (PCR), such as real-time quantitative PCR. However, Eva Green has been proven to restrict PCR to a lesser extent compared to others.
Proteins Gel Stains
The two most commonly used protein stains include Coomassie Brilliant Blue and Silver stains. Coomassie blue stain non-specifically binds to proteins, and, when the excess stain is removed by destaining, you can easily observe blue protein bands. The stain involves the use of two forms of dyes: G-250 (“colloidal”) or the R-250 form. The formulations are highly compatible with downstream applications, such as mass spectrometry, western blotting, and sequencing.
The silver stain is a more sensitive colorimetric method than coomassie staining as it can detect protein even present in a lower quantity. It deposits at the locations of protein bands for their visualization. However, the technique is expensive and can also yield false bands because of its ability to bind polysaccharides and nucleic acids.
There are also some fluorescent dye stains for the total protein that are used for their fast and easy procedures. However, those whose emission and excitation maxima correspond to that of commonly used filter sets and laser settings for fluorescence imaging are the most useful. They are extensively used in downstream applications, like western blotting, 1D and 2D gel electrophoresis, and mass spectrometry.
What is Gel Electrophoresis Stain Used For?
Gel electrophoresis stains are mainly used to visualize and identify proteins and nucleic acids (DNA and RNA) after running agarose or polyacrylamide gels.
The role of stains in visualizing DNA fragments is exploited extensively in labs for a range of purposes. It includes determining the size and quantifying the molecule, confirming the presence of DNA fragments in any given sample for medical or research purposes, and detecting or studying genetic variations or mutations by comparing the size of the stained genetic components.
What Industries Use Gel Electrophoresis Stain?
Because of its crucial role in the visualization of macromolecules, gel electrophoresis stains are used in a wide range of labs and industries related to life science, manufacturing, medicine, or biotechnology.
Gel electrophoresis stain is used in research to analyze nucleic acid and proteins in different samples. Further, it’s also used for criminal investigation, identifying microorganisms, and studying microbial communities.
The pharmaceutical industry uses protein gel electrophoresis stains for protein purification and characterization. For example, it identifies impurities or differences in glycosylation patterns. Further, in medicine, it’s used to identify and study nucleic acids and proteins to identify diseases and develop new treatments.
In the food industry, the gel stain is used to detect and quality the contaminants, such as viruses, bacteria, and others, and their level in different food products. Further, it’s also used to identify food allergens, safeguard the quality of food products, and detect unique protein profiles to authenticate food products.
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Gel electrophoresis stain is a chemical used to visualize and identify separated proteins, DNA, and RNA in a gel. Based on the molecule the stain is used to visualize, it’s categorized into two groups: nucleic acid gel stain and protein gel stain.
Some commonly used staining solutions include ethidium bromide (EtBr), coomassie brilliant blue, silver stain, and SYBR green. A range of life science and pharma industries exploit these chemicals for several purposes, including forensic investigation, research use, quality control, and environmental monitoring.
However, to obtain accurate and meticulous data, one needs high-quality reagents to perform the experiments. It can be made sure by referring to the safety data sheet (SDS) and documents that come with chemicals. You can also refer to sections where companies mention the use of chemicals and their success in many research studies.
Another essential step to obtain quality and accurate results are by using high-throughput equipment. However, not all labs can afford the expensive equipment required for their research. This often impedes the work in the lab or brings it to an end.
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