Electrophoresis
3 Pages
ELECTROPHORESIS
Principle
Electrophoresis is a process distinguishing and isolating different compounds from
each other. It relies on the fact that charged particles (molecules) can migrate in a
medium if the medium is subjected to an electrical current.
Factors affecting the distance of movement
1- Net charge on the molecule:
Particles with negative net charges move toward the anode (positive pole) where as
particles with positive charges migrate toward the cathode (negative pole).
2- Size and shape of the molecule:
Particles of identical net charge will be distinguished from each other by their size.
Heavier molecules will move slower than lighter ones.
3- Strength of the electrical field:
The higher the electrical current voltage the further distance travelled and the faster
the speed of the movement.
4- Supporting medium physical and chemical nature:
Some compounds need special medium, e.g., large polypeptides or proteins are
done in polyacrylamide gel where as nucleotide oligomers are done in agarose and
polyacrylamide gel.
5- Electrophoretic temperature:
Optimal temperature for migration must be used.
Dr.Talaat Mirza
Instrumentation
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Electrophoresis
3 Pages
Application
Electrophoresis could be implemented on many charged molecules. Such molecules
include Amino Acids, Polypeptide Chains, Proteins, nucleotide oligomers, RNA, DNA,
Phosphorus sugars and any other ampholytes (molecules whose net charge depends
on the pH of the surrounding medium). The medium and voltage power might
change from a compound to another depending on the compound chemical nature
and size. The technique is implemented for various uses:
1- The identification of certain molecules.
2- The isolation of a certain molecule.
3- The molecular weight of certain molecules.
In this lecture, Polyacrylamide Gel Electrophoresis (PAGE) and "Hemoglobin
Electrophoresis" are introduced.
Polyacrylamide Gel Electrophoresis (PAGE)
Used routinely in the analysis of single stranded and double stranded DNA.
Polyacrylamide is cross linked with TEMED to form a porous gel, thus allowing
movement of DNA molecules. Separation of DNA is based on size. For example DNA
bands made of 1000-2000 base pairs (bp) can be resolved in 3.5% acrylamide (W/V)
where as bands of 6-100 bp are resolved using a 20% acrylamide (W/V).
Visualization of the bands could be done by adding a dye such as Ethidium Bromide
before or after electrophoresis. Alternatively, radioactively labeled DNA can be
visualized by autoradiography (X-ray film).
The gel could be of a denaturing or non-denaturing property. Denaturing
polyacrylamide gels are used mainly for sequencing of DNA where as non-denaturing
ones are used to detect mutations. An even better and more sensitive technique is
"Denaturing-Gradient Gel Electrophoresis". This technique is sensitive enough to
detect a single base mutation out of a several hundred long base pairs of DNA.
Dr.Talaat Mirza
Instrumentation
Page 2 of 3
Electrophoresis
3 Pages
Hemoglobin Electrophoresis
Principle
Hemoglobin (Hgb) migrates according to net charges of its constituent proteins and
polypeptide chains. Various types of hemoglobin can be distinguished from one
another according to their movements. Some types of hemoglobin might migrate
identically therefore manipulating pH can result in different movements of such
hemoglobins (Hgb's).
Types
1- Cellulose Acetate:
Performed under alkaline pH = 8.4 – 8.6.
2- Citrate Agar:
Performed under acidic pH = 6.0 – 6.2.
3- Globin Chain Electrophoresis:
Globin chains are separated from Hgb allowing individual electrophoresis of the
alpha and non-alpha chains.
4- Isoelecteric Focusing (IEF):
The pH of this technique varies according to the constituents of the molecule. It
could be between 3 and 10.
The first two are used routinely especially in the diagnosis of the common
hemoglobin variants (hemoglobinopathies) and Thalassemia. The latter two are
used in special cases when the first two techniques could not distinguish the
abnormal hemoglobin.
Dr.Talaat Mirza
Instrumentation
Page 3 of 3