Gel electrophoresis is a fundamental laboratory technique used to separate biological molecules like DNA, RNA, and proteins. This method relies on an electric current to move charged molecules through a porous gel. The gel acts as a sieving matrix, allowing molecules to separate based on their size, shape, and charge. The composition of this gel is crucial for effective separation, as it dictates the environment through which the molecules migrate.
The Primary Gel Material: Agarose
Agarose is a commonly used material for creating gels in electrophoresis, particularly for separating larger molecules such as DNA and RNA, and sometimes sizable proteins. It is a natural polysaccharide, meaning it is a long chain of sugar molecules, derived from certain types of seaweed. When agarose powder is heated in a buffer solution and then cooled, it forms a solid, yet flexible, gel.
At a microscopic level, this process creates a complex network of agarose molecules held together by hydrogen bonds, forming tiny pores. These pores act as a molecular sieve, allowing smaller molecules to pass through more easily and quickly than larger ones.
The Other Key Gel Material: Polyacrylamide
Polyacrylamide is another key material for electrophoresis gels, typically used for separating smaller molecules like proteins and very short DNA or RNA fragments. Unlike agarose, polyacrylamide is a synthetic polymer, not derived from natural sources. It is formed through a chemical reaction called polymerization, involving two main monomers: acrylamide and N,N’-methylenebisacrylamide (bis-acrylamide).
During polymerization, these monomers chemically crosslink, creating a highly uniform and stable gel matrix with a precisely controlled pore structure. This controlled porosity makes polyacrylamide gels suitable for high-resolution separations, especially for proteins, where even small differences in size can be resolved. While acrylamide monomers are known to be neurotoxins, the polymerized gel itself is generally considered safe to handle.
Why Different Gels? Understanding Pore Size
The primary reason for using either agarose or polyacrylamide gels in electrophoresis is their ability to control pore size, which is important for the effective separation of molecules based on their size. This process is known as molecular sieving, where molecules navigate through the intricate pore network. Smaller molecules move through the pores more freely and migrate faster, while larger molecules encounter more resistance and move more slowly.
For agarose gels, varying the concentration of agarose directly impacts the average pore size; a higher agarose concentration leads to a denser matrix with smaller pores, which is ideal for separating smaller DNA fragments, for example, from 0.2 to 1 kilobases. Conversely, a lower agarose concentration creates larger pores, allowing for better separation of larger molecules, such as DNA fragments ranging from 5 to 10 kilobases. In polyacrylamide gels, both the total concentration of acrylamide and the ratio of bis-acrylamide (the crosslinker) influence the pore size and the gel’s rigidity. A higher total acrylamide concentration results in smaller pores, while the bis-acrylamide ratio dictates the degree of crosslinking, further refining the pore structure.
Essential Additives and Their Roles
Beyond the primary gel material, several other components are important for successful gel electrophoresis. Buffer solutions, such as Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE) for nucleic acids, and Tris-Glycine for proteins, are mixed into the gel solution and used as running buffers in the electrophoresis tank. These buffers maintain a stable pH and provide the necessary ions to conduct electricity, ensuring consistent migration of charged molecules.
Loading dyes are added to the samples before they are placed into the gel wells. These dyes, often containing bromophenol blue or xylene cyanol, serve multiple purposes: they make the colorless samples visible for easier loading into the small wells and increase the sample’s density, ensuring it sinks into the well rather than diffusing into the buffer. They also contain tracking dyes that migrate through the gel, allowing researchers to monitor the progress of the separation without disturbing the samples.
After electrophoresis, DNA-binding stains like ethidium bromide or SYBR Green, or protein stains such as Coomassie blue or silver stain, are used to visualize the separated molecules. These stains bind to the target molecules and fluoresce under UV light or produce a visible color, revealing the distinct bands of separated molecules.