Electrophoresis is a general term that describes the migration and separation of charged particles under the influence of an electric field.

1. Electrophoresis

2. Definition of Electrophoresis
• Electrophoresis is migration of charged
particles or molecules in a medium under the
influence of an applied electric field.
• A technique for separating the components of
a mixture of charged molecules (proteins,
DNAs, or RNAs) in an electric field within a gel
or other support. The movement of
electrically charged molecules in an electric
field often resulting in their separation.

4. Principles of Electrophoresis
Electrophoresis is a general term that describes the migration
and separation of charged particles (ions) under the influence
of an electric field. An electrophoretic system consists of two
electrodes of opposite charge (anode, cathode), connected by
a conducting medium called an electrolyte. The separation
effect on the ionic particles results from differences in their
velocity (v), which is the product of the particle's mobility (m)
and the field strength (E):
1v=mE.
The mobility (m) of an ionic particle is determined by particle
size, shape, and charge, and the temperature during the
separation, and is constant under defined electrophoretic
conditions

5. Types of Electrophoresis
1. Routine (zone) electrophoresis : The
traditional clinical laboratory electrophoresis
performed on a rectangle-shaped slab gel.
Uses: Separation of proteins.
Some use in separating nucleic acids.

6. 2. High resolution electrophoresis (HRE)
Routine electrophoresis using a high voltage.
Uses: Conditions where more resolution of
proteins is needed, eg, separation of CSF
proteins for the diagnosis of multiple sclerosis
and light chains in urine for early detection of
lymphoproliferative disorders such as multiple
myeloma.

7. 3. Polyacrylamide electrophoresis (PAGE)
Horizontal or vertical slabs or gel incorporated
into vertical cylinders or rods. DNA of 100 base
pairs (bp) or less can be separated.
Uses: Used to study individual proteins in serum,
especially genetic variants and isoenzymes
(polyacrylamide gel may yield 20 or more
fractions)

8. 4. Capillary electrophoresis(CE)
Combines electrophoresis and high performance
liquid chromatography. CE takes place in a very
thin, fused silica capillary tube with
polyacrylamide or agarose gel.
Uses: Commonly used in the clinical laboratory.
Very rapid, efficient, easily automated,
computerized, and requires only a microvolume
of sample.

9. 5. Isoelectric focusing electrophoresis (IEF)
The gel is infused with chemicals that make a pH
gradient across the surface of the gel (ampholytes).
Using very high voltage, proteins will then migrate
to the point on the gel where they have no net
charge, ie, their isoelectric point.
Uses: Separation of variant hemoglobins in prenatal
screening and isoenzymes. Detection of oligoclonal
bands in gamma-globulin

10. 6. Immunofixation electrophoresis
An agarose gel electrophoresis separates the
proteins in a serum sample. Antiserum against the
protein of interest is spread directly on the gel. The
protein of interest precipitates in the gel matrix.
After a wash step to remove other proteins, the
precipitated protein is stained.
Uses: Study protein antigens and their split
products.
Identify proteins found in multiple myeloma

11. 7. Pulsed field electrophoresis
Fragment separation is achieved by alternately
applying the power to different pairs of electrodes.
The most common method alternates the positive
and negative electrodes in cycles during
electrophoresis
Uses: Separates larger fragments of DNA (> 50
kilobases) that cannot be separated with AGE or
PAGE in routine electrophoresis systems.

12. 8. Two-Dimensional electrophoresis
Separating the same sample with two distinct
separation techniques or two different
electrophoresis separations. The separated bands
from one electrophoresis are resolved more with
the second electrophoresis. IEF followed by PAGE or
AGE is the most frequent two-dimensional
electrophoresis. The gel from the IEF capillary is
removed and placed across the PAGE or AGE gel
slab at right angles for the second electrophoresis.

13. Uses: Most applications are in research fields.
Used to study families of proteins in the field of
proteomics and protein content in different
types of cells. Used in genetics to study
differences in diseases, gene mutations, and
bacterial DNA. Used to study tumor cells to
detect malignancies.