Electrophoresis is defined as the separation (migration) of charged particles
through a solution or gel, under the influence of an electrical field.
The rate of movement of particle depends on the following factors.
What is Gel 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. A gel is a colloid in a solid form. The term electrophoresis describes
the migration of charged particles under the influence of an electric field.
“Electro” refers to the energy of electricity. “Phoresis,” from the Greek verb
phoros, means “to carry across.” Thus, gel electrophoresis refers to the technique
in which molecules are forced across a span of gel, motivated by an electrical
current. Activated electrodes at either end of the gel provide the driving force.
A molecule’s properties determine how rapidly an electric field can move the
molecule through a gelatinous medium.
Many important biological molecules such as amino acids, peptides,
proteins, nucleotides, and nucleic acids, possess ionizable groups and, therefore,
at any given pH, exist in solution as electrically charged species, either as
cations (+) or anions (–). Depending on the nature of the net charge, the charged
particles will migrate to either the cathode or the anode.
How does this Technique Work?
Gel electrophoresis is a technique used for the separation of nucleic acids and
proteins. Separation of large (macro) molecules depends upon 2 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 2 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.
There are 2 basic types of materials used to make gels: agarose and
polyacrylamide. Agarose is a natural colloid extracted from seaweed. It is very
fragile and easily destroyed by handling. Agarose gels have very large “pore”
size and are used primarily to separate very large molecules, with a molecular
mass greater than 200 kdal. Agarose gels can be processed faster than
polyacrylamide gels, but their resolution is inferior. That is, the bands formed
in the agarose gels are fuzzy and spread far apart. This is a result of pore size
and cannot be controlled.
Agarose is a linear polysaccharide (average molecular mass about 12,000)
made up of the basic repeat unit agarobiose, which composes alternating units
of galactose and 3,6-anhydrogalactose. Agarose is usually used at concentrations
between 1% and 3%.
Agarose gels are formed by suspending dry agarose in an aqueous buffer,
then boiling the mixture until a clear solution forms. This is poured and allowed
to cool to room temperature to form a rigid gel.
There are 2 basic types of materials used to make gels: agarose and
polyacrylamide. The polyacrylamide gel electrophoresis (PAGE) technique was
introduced by Raymond and Weintraub (1959). Polyacrylamide is the same
material that is used for skin electrodes and in soft contact lenses. Polyacrylamide
gel may be prepared so as to provide a wide variety of electrophoretic conditions.
The pore size of the gel may be varied to produce different molecular seiving
effects for separating proteins of different sizes. In this way, the percentage of
polyacrylamide can be controlled in a given gel. By controlling the percentage
(from 3% to 30%), precise pore sizes can be obtained, usually from 5 to 2000
kdal. This is the ideal range for gene sequencing, protein, polypeptide, and
enzyme analysis. Polyacrylamide gels can be cast in a single percentage or with
varying gradients. Gradient gels provide a continuous decrease in pore size
from the top to the bottom of the gel, resulting in thin bands. Because of this
banding effect, detailed genetic and molecular analysis can be performed on
gradient polyacrylamide gels. Polyacrylamide gels offer greater flexibility and
more sharply defined banding than agarose gels.
Mobility of a molecule =
||(applied voltage) × (net charge of the molecule) /
friction of the molecule (in the electrical field)
v (velocity) =
||E (voltage) × q (charge)/f (frictional coefficient).
Polyacrylamide Gel Electrophoresis
Polyacrylamide is the solid support for electrophoresis when polypeptides, RNA,
or DNA fragments are analyzed. Acrylamide plus N,N’-methylene-bis-acrylamide
in a given percentage and ratio are polymerized in the presence of ammonium
persulfate and TEMED (N,N,N’,N’-tetra-methyl-ethylene-diamine) as catalysts.
Safety and Practical Points
Polyacrylamide Gel Electrophoresis of Proteins
- Acrylamide and bis-acrylamide are toxic as long as they are not
- Buffer (usually Tris) and other ingredients (detergents) are mixed with
acrylamide before polymerization.
- Degassing of acrylamide solution is necessary before pouring the gel
because O2 is a strong inhibitor of the polymerization reaction.
- Under nondenaturing conditions.
- Under denaturing conditions.
- Isoelectric focusing.
These techniques are used to analyze certain properties of a protein such
as: isoelectric point, composition of a protein fraction or complex, purity of a
protein fraction, and size of a protein.
We will concentrate on denaturing polyacrylamide gel electrophoresis in
the presence of sodium dodecylsulfate (SDS-PAGE) and a reducing agent (DTT,
or dithioerithritol, DTE).
The protein is denatured by boiling in “sample buffer,” which contains:
- Buffer pH 6.8 (Tris-HCl).
- DTT or DTE.
- Bromophenol blue (tracking dye).
Discontinuous Polyacrylamide Gel Electrophoresis
FIGURE 1 Electrophoresis.
This type of polyacrylamide gel consists of 2 parts:
The running gel has a higher percentage (usually 10%–15%) of acrylamide
and a Tris-HCl buffer of pH 8.8.
- The larger running (resolving) gel
- The shorter upper stacking gel.
The stacking gel usually contains 5% acrylamide and a Tris-HCl buffer of
The buffer used in SDS-PAGE is Tris-glycine with a pH of about 8.3.
FIGURE 2 Protein passage through a disc-gel electrophoresis system.
Determination of the Molecular Weight of a Polypeptide by SDS-PAGE
Since all polypeptides are wrapped with SDS and thus are strongly negatively
charged, they migrate through the running gel according to their size (small
polypeptides migrate faster than large ones!).
There is a linear relationship between the log of the molecular weight of
the polypeptide and its migration during SDS-PAGE.
Standard polypeptides have to be run on the same gel and a curve of their
migration versus the log of their molecular weight has to be generated.