electrophoresis

Shanes
introduction
• Introduced by TISELIUS in 1937, for the separation of proteins.
• Defined mainly as the migration of charged molecules under the
influence of an external magnetic field.
• for the separation of
• Proteins
• Nucleic acid
• Polysaccharides
Principle
• Rate of migration (separation) depends upon e/m (charge to mass)
ratio
• The migration of particles or the rate of travel of particle, in
electrophoretic system depends on properties of the particles as
well as the instrumental system
1. Characteristic of particles
2. Property of electric field
3. Temperature
4. Nature of suspended medium
5. Buffering system
6. Nature and size of molecules to be separated

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TYPES

• There are a number of different types of

electrophoresis.
• All involve generating an electric field between to
points and placing a matrix of some sort in-between
through which the macromolecules must travel.
• The speed at which they pass through this matrix in
the presence of the electric field is called their
electrophoretic mobility.
DIFFERENT TYPES

• Agarose gel electrophoresis

• DNA denaturing polyacrylamide gels
• Electrofocusing electrophoresis
• Capillary electrophoresis.
• Native PAGE
• SDS-PAGE.
Agarose gel electrophoresis
• a method of separating charged molecules in an electrical field; DNA
has an overall negative charge

• Used to separate DNA fragments by size

Principle
• In electrophoresis, the charged molecules migrate
across support medium [a gel], because they are placed
in an electrical field.

• The gel acts as a sieve to retard the passage of

molecules according to their size and shape.

• The negatively charged particles move toward the

positive electrode while the positive charge particles
move toward the negative electrode.

• Proteins and nucleic acids [because of being charged]

are separated by electrophoresis.
Components of an Electrophoresis System
• Power supply and chamber, a source of negatively
charged particles with a cathode and anode
• Buffer, a fluid mixture of water and ions
• Agarose gel, a porous material that DNA migrates through
• Gel casting materials
• DNA ladder, mixture of DNA fragments of known lengths
• Loading dye, contains a dense material and allows
visualization of DNA migration
• DNA Stain, allows visualizations of DNA fragments after
electrophoresis
 Cathode

-
Anode
+

Buffer
Dyes Power Supply
Agarose
gel
Bio-Rad’s Electrophoresis Equipment

Power Supplies

Precast Ready
Agarose Gel
Electrophoresis Buffer
• TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA) are
the most common buffers for duplex DNA
• Establish pH and provide ions to support conductivity
• Concentration affects DNA migration
• Use of water will produce no migraton
• High buffer conc. could melt the agarose gel

• New Sodium Borate (SB) buffer allows gels to be run

at higher voltages in less time than traditional buffers
 Agarose Gel
• A porous material derived from red
seaweed
• Acts as a sieve for separating DNA
fragments; smaller fragments travel
faster than large fragments 1% agarose

• Plinko Model
• Concentration affects DNA migration
• Low conc. = larger pores better
resolution of larger DNA fragments
2% agarose
• High conc. = smaller pores better
resolution of smaller DNA fragments
Loading Dye
• DNA samples are loaded into
a gel AFTER the tank has been
filled with buffer, covering the
gel
• Contains a dense substance,
such as glycerol, to allow the
sample to "fall" into the
sample wells
• Contains one or two tracking
dyes, which migrate in the gel
and allow monitoring of how
far the electrophoresis has
proceeded.
 DNA Staining
• Allows DNA
visualization after gel
electrophoresis

• Ethidium Bromide

• Bio-Safe DNA stains

• In gel staining
Frequent Misconceptions

Agarose
Gel

DNA
Fragment
s

• Each band on the gel represents a single DNA

strand...NOT TRUE.
• A single band/position in a lane contains only one
type of DNA sequence...NOT ALWAYS TRUE.
An Electrophoretogram
Lane 2:
Uncut Lambda DNA
Lane 3:
Lambda DNA cut with EcoRI
Lane 4:
Lambda DNA cut with HindIII
Lane 5:
Lambda DNA cut with EcoRI and HindIII
Lane 6:
DNA Ladder
APPLICATIONS OF ELECTROPHORESIS

Forensics
Applications contd..
• Electrophoresis is employed in biochemical and clinical field.
• In the study of protein mixtures
• Antigen antibody reactions
• In fractioning protein.
• In analysis of lipoprotein
• Hemoglobin
• In combination with autoradiography
• Separation of organic acid, alkaloids, carbohydrates, amino acids, alcohols,
phenols, nucleic acids, insulin.
• In food industry

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