Electrophoresis is a technique used to separate charged particles in a fluid under an electric field, first observed in 1807. It is utilized in laboratories for analyzing macromolecules like DNA and proteins, with variations such as slab and capillary electrophoresis tailored for different applications. The method relies on the size, charge, and shape of molecules, with factors such as pH and viscosity influencing their mobility in the electric field.

1. ELECTROPHORE
SIS

2. DEFINITION
Electrophoresis is the motion of dispersed particles relative to a
fluid under the influence of a spatially uniform electric
field. This electro-kinetic phenomenon was observed for the
first time in 1807 by Ferdinand Frederic Reuss (Moscow State
University),who noticed that the application of a
constant electric field caused clay particles dispersed in water to
migrate. It is ultimately caused by the presence of a charged
interface between the particle surface and the surrounding
fluid. It is the basis for a number of analytical techniques used
in biochemistry for separating molecules by size, charge, or
binding affinity.

3. ■ Electrophoresis of positively charged particles (cations) is
called Cataphoresis, while electrophoresis of negatively charged
particles (anions) is called Anaphoresis.
■ Electrophoresis is a technique used in laboratories in order to
separate macromolecules based on size. The technique applies a
negative charge so proteins move towards a positive charge. This is
used for both DNA and RNA analysis.
■ Poly-acryl-amide gel electrophoresis (PAGE) has a clearer resolution
than agarose and is more suitable for quantitative analysis.
■ In this technique DNA foot-printing can identify how proteins bind
to DNA. It can be used to separate proteins by size, density and
purity. It can also be used for plasmid analysis, which develops our
understanding of bacteria becoming resistant to antibiotics.

4. PRINCIPLE
■ Biological molecules exist in a solution as electrically charged
particles at a given pH.
anionic (+vely charged/basic) Zwitterions
cationic (-vely charged / acidic) or amphoteric molecule
■ pH greatly influences the total charge of molecules.
■ When electricity is applied to the medium containing
biological molecules, depending on their net charge &
molecular size, they migrate differentially, thus different
proteins/DNA can be separated.

5. ■ The velocity (v) of charged molecule in an electric field-
v = Eq / F
where F = frictional coefficient, which depends upon the mass
and shape of the molecule.
E = electric field (V/ cm)
q = the net charge on molecule
v = velocity of the molecule
■ The value of E depends upon
* The charge of the analyte ion
* the frictional retarding force which indicates:
- size and shape of the ion
- viscosity of the medium in which migration occurs

6. ■ The movement of charged particles in an electric field is expressed in
the terms of electrophoretic mobility, denoted by
where µ= V/E
or µ= q/F
■ For molecules with similar conform F varies with size but not with
shape. Thus electrophoretic mobility (µ)of a molecule is directly
proportional to charge density (charge/mass ratio).
■ In electrophoresis, the charged molecules under the influence of
electric field migrate towards oppositely charged electrode.
■ Those molecules with positive charge move towards cathode and
negatively charged molecules move towards anode.
■ The migration is due to charge on the molecules and potential
applied across the electrodes.

7. ■ The samples under test is placed at one end of the paper near one of
the electrodes when the electricity is applied, the molecules start
moving towards the respective electrodes.
■ But the movement is influenced by molecular weight of the molecule.
So when a mixture is placed on the electrophoresis paper or gel,
different bands are seen along the paper after the process. This is
due to differential rate of movement by molecules based on their
weight.
■ Those molecules with higher molecular weight move slower. While
those with small weight move faster. Also the size of a molecule also
influences the movement.
■ The bigger size molecules experience more friction than smaller ones
in motion.
■ Those molecules migrate at different speed and to different lengths
based on their charge, mass and shape.

10. Factors affecting
Electrophoresis
Electrophoretic mobility depends on :
■ 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.
Therefore, globular proteins move faster than fibrous ones.

11. Electrophoretic velocity depends on:
Inherent factors:
■ Magnitude of charge
■ Charge density
■ Molecular weight
■ 3° or 4° structure
Coherent factors:
■ Solution pH
■ Electric field
■ Solution viscosity
■ Temperature

12. TYPES OF
ELECTROPHORES
IS
AND THEIR
TECHNIQUES

13. ■ Electrophoresis can broadly be divided into two types:
■ Slab Electrophoresis
■ Capillary Electrophoresis

14. SLAB ELECTROPHORESIS
■ Method mostly used for industrial scale
■ Its slow, time consuming and bulky.
■ Sole method available for separation of proteins, enzymes,
hormones, antibodies, nucleotides like DNA and RNA.
■ Process- simple, difficult to automate, poor quantitation,
large quantities, large cross-sectional area, short length
leading to low electrical resistance, high currents.
■ Uses flat gels formed between two glass plates.
■ 2D analysis

15. ■ Running of multiple samples simultaneously in same gel.
■ Gels are with multiple ‘lanes’ such that samples run parallel, size and
number of lanes can be varied and since they run in same medium,
there is less likelihood of sample variation due to minor changes in
gel structure.
■ Used mostly for Blot analysis and Auto Radiographic analysis.
■ This method is a standard for routine nucleic acid analysis, and
those employing antigenic controls.

16. Apparatus Setup For Slab Gel
Electrophoresis

17. ■ This slab electrophoresis is further divided into three types based on
the principle used for separation.
a. Zone Electrophoresis
b. Iso electric- Focusing
c. Immune-Electrophoresis

18. ZONE ELECTROPHORESIS
■ Here, the charged particles are separated into different zones
or bands in a buffer and stabilized in solid porous or any
other support medium.
■ Ex: filter paper, agar gel, or poly acryl amide gel.
■ This is of two types:
1. Paper Electrophoresis
2. Gel Electrophoresis

19. 1. Paper Electrophoresis:
■ Employs Whatmann Filter Paper No.1 which is moistened by a buffer
and then connected at two ends to two points charged electrodes.
■ Sample is applied to one end and left for separation of components
under electric field.
■ After separation, the paper is dried and stained to get coloured
bands.
■ The coloured bands are recognized for nature of sample by
comparing with the standard.
■ For a sample of serum, 5 bands of proteins can be separated by
paper electrophoresis.

20. ■ This is commonly used method for analysis and resolution of small
molecules.
■ Sample is applied on a strip of filter paper wetted with desired buffer
solution.
■ The ends of the strip are dipped into the buffer reservoirs in which
the electrodes are placed.
■ The electric current is applied allowing the molecules to migrate for
sufficient time.
■ Paper is removed and dried and stained to be detected.
■ Coloured can be identified by comparing with a set of standards run
simultaneously.

21. ■ For separation of serum proteins, Whatmann filter paper No.1,
veronal or tris buffer at pH 8.6 and the strains amido black or
bromo-phenol blue are employed.
■ Serum proteins are separated into 5 distinct bands- albumin, α 1-, α
2-, β-, γ- globulins.

24. 2. Gel Electrophoresis:
■ This involves separation of molecules based on their size, in addition
to the electrical charges.
■ Movement of large molecules is slow, resolution is much higher.
■ Thus, the serum proteins can be separated to about 15 bands, instead
of 5 bands on paper electrophoresis.
■ Gels commonly used in gel electrophoresis are agarose and poly acryl
amide, sodium dodecyl sulphate (SDS).
■ Poly acryl amide is employed for the determination of molecular
weights of proteins in a popularly known technique known as “ SDS-
PAGE”.
■ More efficient than paper type as the rate of movement is slow and
area of separation is larger by thickness.

25. ■ Sample is applied and subjected to electric field which can lead to
separation of molecules. They form a band and can be recognized by
staining and comparing with standard sample bands.

26. ISO ELECTRIC FOCUSING
■ Based primarily on immobilization of molecules at iso
electric pH during electrophoresis.
■ Iso electric pH is set at different foci and hence, the molecles
are immobilized at their iso-electric point.
■ They don’t move towards the electrodes, but stay at their iso
electric pH.
■ Very efficient to separate the proteins and from serum,
almost 40 bands of proteins can be fromed.

27. ■ 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, migrate to positions
corresponding to their iso-electric points, get immobilized and form
sharp stationary bonds.
■ Gel blocks can be stained and identified.
■ Iso-electric focusing can be conveniently used for the purification of
proteins.

28. IMMUNO
ELECTROPHORESIS
■ 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.
■ 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.