Polyacrylamide gel electrophoresis (PAGE) separates molecules based on size and charge. Factors like net charge, size, strength of the electrical field, and temperature affect how far molecules migrate through the gel. PAGE is used to identify and isolate molecules like proteins, DNA, and hemoglobin variants. For example, hemoglobin electrophoresis separates different hemoglobin types based on their net charge and migration through gels of varying pH.

1. Polyacrylamide Gel
Electrophoresis
Scott Malcolm

2.  Principle
 Factors affecting the distance of movement
 Application
 Polyacrylamide Gel Electrophoresis (PAGE)
 Hemoglobin Electrophoresis
Objectives

3. 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.
Electrophoresis

5. 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.
Factors affecting the distance of movement

6.  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.
Application

7.  1- The identification of certain molecules.
 2- The isolation of a certain molecule.
 3- The molecular weight of certain molecules.
The technique is implemented for various uses:

8. Polyacrylamide Gel
Electrophoresis (PAGE)

9.  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).

10.  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.

11. 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).
Hemoglobin Electrophoresis

12. 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.
Types

14. Thank you