Electrophoresis is a fundamental laboratory technique used across molecular biology and biochemistry to analyze complex mixtures of molecules. This process uses an electrical field to move charged macromolecules, such as DNA, RNA, and proteins, through a porous matrix. The matrix acts like a molecular sieve, separating molecules primarily based on their size and shape. Agarose and polyacrylamide are the two most common materials used to form these separation matrices, each suitable for different analytical tasks.
Fundamental Composition and Gel Formation
Agarose is a naturally occurring polysaccharide derived from red seaweed. The gel matrix forms when agarose powder is dissolved in a buffer solution at boiling temperatures and then allowed to cool. This cooling process facilitates the formation of a three-dimensional network through non-covalent hydrogen bonding between the helical agarose chains. The resulting matrix is a relatively rigid, porous mesh that is simple to prepare and generally non-toxic.
In contrast, the polyacrylamide gel matrix is a synthetic polymer formed through vinyl addition polymerization. The process involves the copolymerization of acrylamide monomers with a cross-linking agent, typically N, N’-methylenebisacrylamide (bis-acrylamide). This reaction requires two catalysts: ammonium persulfate (APS), which acts as the free-radical initiator, and tetramethylethylenediamine (TEMED), which accelerates the rate of free-radical generation. This chemical synthesis creates a stable, chemically cross-linked hydrogel network. The unpolymerized acrylamide monomer is a known neurotoxin, necessitating careful handling and safety precautions during preparation.
Separation Scope and Target Molecules
The distinct physical structures of the two gels dictate their primary applications. Agarose gels are characterized by large, random pore sizes, making them the preferred medium for separating large macromolecules, particularly nucleic acids. They are routinely used to analyze DNA fragments ranging from approximately 100 base pairs (bp) up to 50 kilobase pairs (kb), and even megabases with specialized techniques. Typical applications include verifying the size of polymerase chain reaction (PCR) products, separating genomic DNA digests, and isolating plasmid DNA.
Polyacrylamide gels, with their finer and more uniform pore structure, are optimized for resolving smaller molecules. They are the standard choice for protein separation, often performed in the presence of sodium dodecyl sulfate (SDS-PAGE). Polyacrylamide is also used for analyzing small nucleic acid fragments, such as oligonucleotides, microRNAs, and DNA fragments generally less than 1 kilobase pair. Its high resolving power is necessary for applications like DNA sequencing, where the separation of fragments differing by only a single base pair is required.
Resolution and Pore Size Control
The difference between the two matrices lies in the degree of control over the pore size and the resultant separation resolution. In an agarose gel, the pore size is controlled indirectly by adjusting the total concentration of the agarose powder in the buffer solution. A higher percentage of agarose leads to a denser network with smaller average pores, suitable for resolving smaller DNA fragments. However, the pore geometry is inherently less uniform and more random, limiting the gel’s ability to precisely differentiate molecules that are similar in size.
Polyacrylamide offers superior resolution because its matrix structure can be precisely tuned. The pore size is controlled by adjusting two independent variables: the total concentration of acrylamide and cross-linker (T) and the ratio of cross-linker to monomer (C). Varying these concentrations allows researchers to optimize the pore size for a specific range of molecular weights. This yields high resolving power capable of separating molecules that differ by only a few amino acids or a single nucleotide. A gel can also be cast with a concentration gradient, featuring a low percentage at the top and a high percentage at the bottom, which allows a wide range of molecular sizes to be separated effectively on a single gel.
Practical Considerations and Safety
Preparation and handling procedures differ significantly between the two gel types. Agarose gels are typically cast horizontally in an open tray and run submerged in buffer, which simplifies the casting process. The solidified agarose is relatively stable and easy to handle, although low-concentration gels can be physically fragile. The preparation process is rapid, involving only heating and cooling.
Polyacrylamide gel casting is more technically demanding, often requiring specialized vertical casting apparatus with glass plates. This method allows the gel to be run vertically, necessary for the high-resolution separation of proteins and small nucleic acids. The chemical polymerization step must be performed quickly after adding the catalysts, as the reaction proceeds spontaneously. A primary safety consideration is the neurotoxic nature of the unpolymerized acrylamide monomer. This mandates the use of proper personal protective equipment and a fume hood during preparation, a concern absent when handling solidified agarose.