Electrophoresis is a method frequently used in biochemistry and molecular biology in order to separate macromolecules, usually proteins or nucleic acids. This separation allows for individual proteins or nucleic acid sequences to be isolated and analyzed from a complex mixture of them. A typical example of its use would be in separating DNA fragments that had been produced from a microbial community using PCR (Polymerase Chain Reaction).
Principles of Electrophoresis
Electrophoresis separates molecules along a gradient based on their size, charge or properties. That gradient can be an electrical field or, in the case of Denaturing Gradient Gel Electrophoresis (DGGE), a denaturant such as a mixture of urea and formamide. Proteins will migrate toward the anode if negatively charged and the cathode if positively charged; the distance of migration depending on the size.
Denaturing Gradient Gel Electrophoresis
With DGGE, DNA moves along a gradient of increasing denaturing power, until the power is sufficient to denature that particular DNA fragment. At this point the migration stops, meaning that fragments will be separated based on their susceptibility to denaturing.
What the Buffer Does
In the case of electrophoresis that separates on the basis of charge, the charge is transmitted via the ions provided by the buffer. The buffer, by providing a reservoir of weak acid and base, also keeps the pH within a narrow range. This is important because the structure and charge of a protein or nucleic acid will change if subjected to significant pH changes, thus preventing proper separation.
Different buffers work best for different desired pH ranges. Typical buffers used include acetic, boric, phosphoric and citric acids, glycine and taurine. Generally, the pKa value (acid dissociation constant) should be close to the required pH, and buffers providing species of low charge magnitude are preferred, so as not to conduct too much current.
Electrophoresis separates macromolecules by size, charge and/or properties, and a buffer is required to provide a stable pH and conduction of charge.