Gel electrophoresis is a fundamental laboratory technique that separates biological molecules, such as DNA, RNA, and proteins, based on their size and electrical charge. This process involves moving these molecules through a gel matrix using an electric current. However, after this separation occurs, the molecules remain invisible to the naked eye. Consequently, a subsequent step of staining is essential to make these separated molecules detectable for analysis.
Why Molecules in Gels Are Invisible
Biological molecules like DNA, RNA, and proteins are microscopic, too small to be observed as distinct bands within a gel. They are imperceptible to the naked eye.
The gel itself is a translucent, jelly-like substance. It provides a clear, porous medium through which the molecules migrate during electrophoresis, but it offers no inherent contrast to the invisible biological samples embedded within it. Without a method to highlight their positions, the separated molecules would simply blend into the clear gel. This renders the electrophoresis procedure impractical for scientific analysis without a visualization step.
How Stains Visualize Molecules
Stains are chemical compounds designed to bind to target molecules within the gel, making them visible. These compounds possess properties that allow for their detection, often through light absorption or emission. The general principle involves the stain interacting with the separated molecules, thereby creating a localized signal that can be observed.
For nucleic acids like DNA and RNA, fluorescent intercalating dyes are commonly used. These dyes, such as Ethidium Bromide (EtBr), insert themselves between the base pairs of the nucleic acid double helix. When exposed to ultraviolet (UV) light, the bound dye fluoresces, emitting visible light, typically an orange color, allowing the DNA or RNA bands to be seen.
While EtBr has been historically used due to its strong fluorescence and affordability, it is a known mutagen. Safer alternatives have been developed, including SYBR Green and GelRed, which also fluoresce under UV or blue light but pose less of a health risk. These stains can be incorporated into the gel before electrophoresis or applied by soaking the gel afterward.
Protein visualization often employs different types of stains. Coomassie Brilliant Blue is a widely used general protein stain that binds non-specifically to amino acid residues in proteins, producing a visible blue color. For more sensitive detection of smaller protein quantities, silver staining is employed. This method involves silver ions binding to proteins, which are then chemically reduced to metallic silver, resulting in dark brown or black bands. Fluorescent protein stains, like SYPRO Ruby, also exist, which fluoresce upon binding to proteins.
What Stained Gels Reveal
Once stained, the visible bands on a gel represent distinct populations of molecules that have separated based on their migration through the gel matrix. This visualization provides several pieces of information. One primary revelation is molecular size; by comparing the migration distance of an unknown band to a molecular weight ladder (a set of molecules of known sizes run in a separate lane), the approximate size of the molecule in question can be estimated. Smaller molecules travel further through the gel than larger ones.
The intensity or brightness of a stained band often correlates with the quantity or abundance of the molecule present in that band. A darker or brighter band generally indicates a higher concentration of the molecule, enabling semi-quantitative analysis. Stained gels also confirm the presence or absence of a specific molecule in a sample. They can also assess the purity of a purified sample; a single, well-defined band typically indicates a highly pure sample. This information finds broad applications across scientific disciplines, from confirming successful DNA amplification in research to identifying specific proteins in diagnostic tests, and even in forensic analysis for DNA fingerprinting.