Gel electrophoresis is a laboratory technique used to separate large biological molecules, such as DNA, RNA, and proteins, based on their physical properties. This method relies on an electric field to move charged molecules through a porous gel matrix. The technique allows for the analysis and purification of these molecules by sorting them primarily by size, but also by charge and shape. Understanding the system’s components provides insight into how this separation is achieved and why it’s a widely used tool in molecular biology and biochemistry.
The Core Medium: The Gel Itself
The gel is the central element of gel electrophoresis, functioning as a sieving medium. This porous matrix allows molecules to migrate through it when an electric current is applied. The two primary materials used to create these gels are agarose and polyacrylamide, each selected for different applications based on the size of the molecules to be separated.
Agarose
Agarose, a natural polysaccharide derived from seaweed, forms gels with relatively large pores. Its pore size can be adjusted by varying the concentration of agarose in the gel; a higher concentration results in smaller pores. This characteristic makes agarose gels suitable for separating larger molecules, such as DNA fragments ranging from hundreds to tens of thousands of base pairs, and large protein complexes.
Polyacrylamide
Polyacrylamide, in contrast, is a synthetic polymer. These gels produce a highly uniform matrix with much smaller, more controllable pore sizes compared to agarose. This fine pore structure provides superior resolving power, making polyacrylamide gels the preferred choice for separating smaller molecules, including proteins and small DNA or RNA fragments, typically ranging from a few to several hundred base pairs.
The Essential Liquid Environment: Buffers and Stains
Beyond the gel matrix, specific liquid components are essential for the gel electrophoresis process and for visualizing the separated molecules. The gel is immersed in an electrophoresis buffer, which conducts the electric current and helps maintain a stable pH environment throughout the run. Common buffers like Tris-borate-EDTA (TBE) or Tris-acetate-EDTA (TAE) are important for optimal molecule migration and stability within the gel.
Loading Dye
Before loading samples into the gel, they are typically mixed with a loading dye. This dye serves multiple purposes: it makes the colorless sample visible, increases the sample’s density to ensure it sinks into the wells, and contains tracking dyes that migrate through the gel, allowing monitoring of the separation progress. Common tracking dyes include bromophenol blue and xylene cyanol FF.
Stains for Visualization
After separation, the molecules in the gel need to be visualized, as they are otherwise invisible to the naked eye. For DNA and RNA, fluorescent dyes like ethidium bromide (EtBr) or SYBR Safe are commonly used. Ethidium bromide inserts itself between the base pairs of double-stranded DNA and fluoresces under ultraviolet (UV) light, making the DNA bands visible. SYBR Safe also binds to DNA and emits green light when excited by UV or blue light, offering a less hazardous alternative to ethidium bromide.
For proteins, common stains include Coomassie blue and silver stain. Coomassie blue binds to proteins, turning them blue. This method is sensitive enough to detect proteins in the microgram range. Silver staining is a more sensitive technique where silver ions bind to proteins and are then reduced to metallic silver, creating dark bands. This process achieves high detection sensitivity, often capable of visualizing nanogram quantities of protein.
The Supporting Apparatus: Equipment Components
The physical components of a gel electrophoresis setup provide the necessary environment and forces for the separation to occur. The electrophoresis chamber, or tank, is a container designed to hold the gel and the running buffer. It typically features electrodes at either end to establish the electric field across the gel. DNA electrophoresis often uses horizontal tanks, while protein electrophoresis frequently employs vertical tanks.
Power Supply
A power supply is connected to the electrophoresis chamber and is responsible for generating the electric field. It provides a controlled voltage or current to drive the charged molecules through the gel. Without the power supply, the charged molecules would not migrate, and no separation would occur.
Gel Comb
Before the gel solidifies, a comb is inserted into the molten gel material. Once the gel sets, the comb is carefully removed, leaving behind small indentations known as wells. These wells serve as the loading points where the samples containing the biological molecules are pipetted before the electric current is applied. The comb is therefore necessary for creating the precise slots needed to introduce samples into the gel matrix.