Electrophoresis
Gel electrophoresis is a method that separates macromolecules-either nucleic
acids or proteins-on the basis of size, electric charge, and other physical properties.
Separation of large (macro) molecules depends upon two forces: charge and mass. When
a biological sample, such as proteins or DNA, is mixed in a buffer solution and applied to
a gel, these two forces act together. The electrical current from one electrode repels the
molecules while the other electrode simultaneously attracts the molecules. The frictional
force of the gel material acts as a "molecular sieve," separating the molecules by size.
During electrophoresis, macromolecules are forced to move through the pores when the
electrical current is applied. Their rate of migration through the electric field depends on
the strength of the field, size and shape of the molecules, relative hydrophobicity of the
samples, and on the ionic strength and temperature of the buffer in which the molecules
are moving. After staining, the separated macromolecules in each lane can be seen in a
series of bands spread from one end of the gel to the other.
Proteins are amphoteric compounds; their net charge therefore is determined by
the pH of the medium in which they are suspended. In a solution with a pH above its
isoelectric point, a protein has a net negative charge and migrates towards the anode in an
electrical field. Below its isoelectric point, the protein is positively charged and migrates
towards the cathode. The net charge carried by a protein differs from protein to protein.
At a given pH therefore, and under non-denaturing conditions, the electrophoretic
separation of proteins is determined by both size and charge of the molecules.
Nucleic acids however, remain negative at any pH used for electrophoresis and in
addition carry a fixed negative charge per unit length of molecule, provided by the PO4
group of each nucleotide of the nucleic acid. Electrophoretic separation of nucleic acids
therefore is strictly according to .size.
Support matrices: Generally the sample is run in a support matrix such as cellulose
acetate, starch gel, agarose or polyacrylamide gel. The most commonly used support
matrices, agarose and polyacrylamide, which provide a means of separating molecules by
size, in that they are porous gels. A porous gel may act as a sieve by retarding, or in some
cases completely obstructing, the movement of large macromolecules while allowing
smaller molecules to migrate freely. Agarose is used to separate larger macromolecules
such as nucleic acids, large proteins and protein complexes.
Polyacrylamide gel electrophoresis allows protein separation on non-reactive matrix
formed by acrylamide and N, N’methylene bisacrylamide co-monomers that undergo free
radical mediated polymerization to form a meshwork of pores that satirically resist
protein migration. Polymerization is initiated by ammonium persulfate (radical source)
and catalyzed by TEMED (a free radical donor and acceptor). Polyacrylamide, which is
easy to handle and to make at higher concentrations, is used to separate most proteins and
small oligonucleotides that require a small gel pore size.