Electrophoresis is the movement of charged particles in an electrical field, first characterised in solutions by F.F. Reuss. In short, the higher the voltage, the lower the viscosity of the medium; the smaller the charged particle and the greater the charge on that particle the faster it will move. This is used in microbiology to determine the size of unknown particles by making all variables constant, so that the size becomes the sole determining factor in how fast the particle moves.
In gel electrophoresis a solution of large biological molecules (DNA, RNA or protein of unknown size and/or composition) that have been specially prepared is injected into a small well on a gel ‘plate’. On the application of an electical field the particles will migrate through gel and become seperated by size; smaller molecules will move faster through the gel. The unknown solution is ‘run’ with a solution or solutions containing particles of a known size. This allows comparison and primary analysis of the size of the unknown particles.
This technique relies on the porous nature of the gels used, and requires that they be inert. The ability to control the size of the pores allows the gels to be applied to wide range of molecule sizes, and combined with different and variable voltages the technique can be pushed even further. There are two kinds of gel that are used, and both have very specific applications.
The first kind of gel used was polyacrylamide, which consists of long chains of acrylamide monomer combined with a crosslinking protein. This forms a very stable structure with the pore size controlled by the concentration in the gel. It was once the only gel available, but has been succeeded by agarose gel for most DNA anaylsis. Agarose consists of long molecules (polysaccharides) derived from seaweed which form a mesh with pore size dependent on concentration of agar polysaccharides in the gel.
Agarose gel is used to analyse DNA and RNA molecules. They are innately negatively charged and require only minimal preparation for analysis. They are treated with an alkali which disrupts the formation of complex structure, ensuring that their rate of travel is a product of their size, not their structure. They are analysed in a gel with concentration (pore size) suitable for the size of DNA fragment being analysed. For example, very short fragments will be resolved in 2% or even 3% gels, which cause very slow but very clear migration , reffered to as good resolution. 0.7% is the lowest usual limit of gel concentration as they become very weak. Low percentage gels allow fast mobility, but much lower detail (resolution).
For very small DNA fragments and proteins, polyacrylamide gel (PAG) is used. I will focus on the analysis of proteins. Unlike DNA, proteins do not have an innate single charge and therefore require special preparation. Firstly the protein is denatured, like DNA, but using alkali and a substance that breaks sulphur bridges to produce a linear protein, which stops the structure of the protein affecting mobility. The solution is incubated with SDS, a detergent with an innate negative charge which coats the protein, masking the charges of the amino acids. This is then loaded into a PAG and exposed to an electric field as in the agarose gel electrophoresis (AGE). However, the structure of the gel is not as simple as in AGE. Here the gel consists of 2 tiers, the stacking region and the resolving region. The explaination of these layers is not simple, and therefore I will not attempt to explain them here, it is sufficient to know that they are different.
Both the techniques require the samples to be dyed to visualise their progress through the gel, however to actually view the final products verious visualising agents are used. For DNA samples, a substance called ethidium bromide is used. It interposes itself into the double helix structure. It fluoresces on exposure to UV light, allowing visualisation in a uv box or through UV photography. Proteins can be visualised through various dying procedures. The ‘classic’ dye is Coomassie blue, which binds irreversibly to the proteins, producing distinct blue lines.
