Transcript Electrophoresisx

Ayat Zawawi
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Principle
Factors affecting the distance of movement
Application
 Polyacrylamide
Gel Electrophoresis (PAGE)
 Hemoglobin Electrophoresis
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.
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.
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.
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1- The identification of certain molecules.
2- The isolation of a certain molecule.
3- The molecular weight of certain
molecules.
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).
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The gel could be of a denaturing or nondenaturing property.
Denaturing polyacrylamide gels are used
mainly for sequencing of DNA where as nondenaturing 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.
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).
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.
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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.