ELECTROPHORESIS - HEMALA DEVI S

1. By: S.Hemala Devi

2. INTRODUCTION:
Electrophoresis is a physical method of analysis
which involves separation of the compounds that are
capable of acquiring electric charge in conducting
electrodes.
DEFINITION:
 Electrophoresis is the motion of dispersed
particles relative to a fluid under the influence of
a spatially uniform electric field.
 Electrophoresis of positively charged particles
(cations) is sometimes called cataphoresis.

4.  Electrophoresis of negatively charged particles
(anions) is sometimes called anaphoresis.
 The electrokinetic phenomenon of electrophoresis
was observed for the first time in 1807 by Russian
professors Peter Ivanovich Strakhov and Ferdinand
Fredric Reuss at Moscow State University ,who
noticed that the application of a constant electric
field caused clay particlees dispersed in water to
migrate .
 It is ultimately caused by the presence of a charged
interface between the particle surface and the
surrounding fluid.

5.  It is the basis for analytical techniques used in
chemistry for separating molecules by size, charge,
or binding affinity .
 Electrophoresis is used in laboratories to separate
macromolecules based on size .
 The technique applies a negative charge so proteins
move towards a positive charge .
 Electrophoresis is used extensively in DNA ,RNA and
protein analysis .

6. PRINCIPLE:
 Electrophoresis is general term that involves the
migration and separation of charged ions under the
influence of electric current .
 It consists of two electrode anode and cathode and
electrolyte which serve as conducting medium .
IMPORTANCE OF ELECTROPHORESIS:
 It is used to separate macromolecules like DNA , RNA
and proteins .
 DNA fragments are separated according to their
size.

7.  Proteins can be separated according to their size
and their charge.
APPLICATIONS:
 To study homogenecity of a macromolecular
system.
 Analysis of complex biological mixtures .
 Separation of organic acid, alkaloids,
carbohydrates, amino acids, alcohols, phenols,
nucleic acids and insulin.
 Estimation of the DNA molecule.

8.  It is also used for the separation of vitamins.
 Electrophoresis in combination with
autoradiography is used to study the binding of
iron to serum proteins.
ADVANTAGES:
 High separation efficiency .
 Short analysis time .
 Low sample and electrolyte consumption .
 Low waste generation.
 Ease of operation.

9. DISADVANTAGES:
 Gel preparation and casting – exacting n time
consuming.
 Complete reproducibility of gel preparation not
possible.
 Elaborate optical system are required.
 Due to small diameter of the capillary tube ,
heat is dissipated that causes increased
diffusion. Due to this the resolution is not
always proper.

10. FACTORS AFFECTING ELECTROPHORESIS:
 Electrophoretic mobility depends on charge, size
and shape.
 Charge: higher the charge greater the
electrophoretic mobility.
 Size: bigger the molecule, greater are the frictional
and electrostatic forces exerted on it by the
medium .Consequently , larger particles have
smaller electrophoretic mobility compared to
smaller particles.
 Shape: rounded contours elicit lesser frictional and
electrostatic retardation compared to sharp
contours .

11.  Therefore, globular proteins move faster than
fibrous ones.
 Electrophoresis can broadly be divided two
types:
 Slab Electrophoresis
 Capillary Electrophoresis

12. SLAB ELETROPHORESIS :
 Slab – gel electrophoresis is the predominant
technique for the separation of peptides,
proteins , and polynucleotides.
 The slab - gel format provides mechanical
stability for the separation , reduces solute
dispersion from convection and diffusion , and
permits handling for detection , scanning ,
storage .

14.  It is primary method used in clinical
chemistry lab.
 It has ability to simultaneously separate
several samples in one run.
 It uses a rectangular gel regardless of
thickness .
 Gels are cast on sheets of plastic backing.
 It is useful in separation of serum proteins ,
iso-enzymes, lipoproteins , haemoglobin and
fragments of DNA and RNA.

15.  The slab electrophoresis is further divided into
three types based on the principle used for
separation.
Zone Electrophoresis
Iso electric - Focusing
Immune - Electrophoresis

16. ZONE ELECTROPHORESIS:
 Here, the charged particles are separated into
different zones or band in a buffer and
stabilized in solid porous or any other support
medium.
 Ex : Agar gel, Filter paper or Poly acryl amide gel.
 This is of two types :
Paper Electrophoresis
Gel Electrophoresis

17. PAPER ELECTROPHORESIS:

18.  This technique is useful for the seperation of
small charged molecules such as amino acids
and small proteins .
 A drop of sample is applied in a band to a
thin sheet of supporting material , like paper ,
that has been soaked in a slightly – alkaline
salt solution .
 Paper electrophoresis employs filter paper
strips soaked in buffer solution , usually
diethylbarbituric acid and barbituric acid
dissolved in alkali (Veronal buffer),pH 8.6 .

19.  A small volume of serum is placed on the
paper and a direct current passed for several
hours .
GEL ELECTROPHORESIS:

20.  Gel electrophoresis is a method for separation and
analysis of macromolecules and their fragments ,
based on their size and charge .
 It is used in the clinical chemistry to separate
proteins by charge or size and in biochemistry and
molecular biology to separate a mixed population of
DNA and RNA fragments by length , to estimate the
size of DNA and RNA fragments or to separate
proteins by charge .
 Nucleic acid molecules are separated by applying an
electric field to move the negatively charged
molecules through a matrix of agarose or other
substances.

21.  Shorter molecules move faster and migrate than
longer ones because shorter molecules migrate
more easily through the pores of the gel.
 This phenomenon is called sieving .
 Proteins are separated by charge in agarose
because the pores of the gel are too large to
sieve proteins .
 Gel electrophoresis can also be used for
separation of nano particles.

22. ISO ELECTRIC FOCUSING:
 Based primarily on immobilization of
molecules at isoelectric pH during
electrophoresis.
 Isoelectric pH is set at different foci and
hence , the molecules are immobilized at their
iso – electric point .
 They don’t move towards the electrodes , but
stay at their iso-electric pH .

23.  Very efficient to separate the proteins and from
serum, almost 40 bands of proteins can be
formed.
Stable pH gradients are set up, usually in gel,
covering the pH range to include the iso-
electric points of components in a mixture .
As electrophoresis occurs, molecules, migrates to
positions corresponding to their iso-electric
points, get immobilized and form sharp
stationary bonds.

24. Gel blocks can be stained and identified.
Iso-electric focusing can be conveniently used
for the purification of proteins.
IMMUNO ELECTROPHORSIS:
 Involves combination of the principles of
electrophoresis and immunological reactions .
 First proteins are separated on to the
electrophoresis paper, then, the antibodies are
allowed to diffuse through the paper and react
with separated protein molecules in bands.

25.  Useful for analysis of antigens and antibodies.
 The antibodies diffuse and when they come in
contact with antigens, precipitation occurs ,
resulting in the formation of precipitin bands.
CAPILLARY ELECTROPHORESIS:

26.  Technique first described by – Jorgensen and lukacs
(1980’s) .
 It is also referred as :
 High performance capillary electrophoresis
(HPCE)
 Capillary zone electrophoresis(CZE)
 Free solution capillary electrophoresis (FSCE)
 Capillary gel electrophoresis(CGE)
 Capillary isoeletric focusing ( CIEF )
 Micellar electro kinetic chromatography(MEKC)

27.  Used for size and shape separation .
 Separation based on differences in solute size.
It is used for protein analysis.
Detection is by UV absorbance of chromohore.
It is used for DNA sequencing.