Electrophoresis is a technique used to separate charged molecules such as proteins and nucleic acids based on their size and charge. It works by applying an electric field to encourage migration of molecules toward the positive or negative electrode, depending on their charge. There are several types of electrophoresis that use different supporting media like agarose gel, polyacrylamide gel, paper, or capillaries to separate molecules. Factors like voltage, temperature, buffer composition affect separation efficiency during electrophoresis.

1. Electrophoresis
BY : SOORAJ S NAIR

2. ELECTROPHORESIS
• The term electrophoresis describes the migration of
a charged particle under the influence of an electric
field.
• Many important biological molecules, such as amino
acids, peptides, proteins, nucleotides and nucleic
acids, possess ionisable groups and, therefore, at any
pH, exist in solution as electrically charged species
either as cations (+) or anions (-).
• Under the influence of an electric field charged
particles will migrate either to the cathode or to the
anode, depending on the nature of their net charge.

3. Principle of electrophoresis:
• When a potential difference is applied, the molecules with different
overall charge will begin to separate owing to their different
electrophoretic mobility. Even the molecules with similar charge will
begins to separate if they have different molecular sizes, since they will
experience different frictional forces. Therefore, some form of
electrophoresis rely almost totally on the different charges on the
molecules for separation while some other form exploits difference in
size (molecular size) of molecules.
• Electrophoresis is regarded as incomplete form of electrolysis because
the electric field is removed before the molecules in the samples
reaches the electrode but the molecules will have been already
separated according to their electrophoretic mobilities.
• The separated samples are then located by staining with an appropriate
dye or by autoradiography, if the sample is radiolabeled.

4. Factor affecting electrophoresis:
I Nature of charge:
• Under the influence of an electric field these charged
particles will migrate either to cathode or anode depending
on the nature of their net charge.
ii. Voltage:
• When a potential difference (voltage) is applied across the
electrodes, it generates a potential gradient (E), which is
the applied voltage (v) divided by the distance “d” between
the two electrodes i.e. p.d. (E) = V/d.
• When this potential gradient ‘E’ is applied, the force as the
molecule bearing a charge of ‘q’ coulombs is ‘Eq’ Newtons.
• It is this force that drives the molecule towards the
electrodes.

5. • iii. Frictional force
• There is also a frictional force that retards the movement of this
charged molecule.
• This frictional force is the measure of the hydrodynamic size of the
molecule, the shape of the molecule, the pore size of the medium
in which the electrophoresis is taking place and the viscosity of the
buffer.
• The velocity ‘v’ of the charged molecule is an electric field is
therefore given by the equation. U=Eq/f, where ‘f’= frictional
coefficient
• iv. Electrophoretic mobility:
• More commonly a term electrophoretic mobility ( ) of an ion is
used, which is the ratio of the velocity of the ion and the field
strength. i.e. =U/E.
• When a p.d. is applied, the molecule with different overall charges
will begin to separate owing to their different electrophoretic
mobility.
• Even the molecule with similar charges will begin to separate if they
have different molecular sizes, since they will experience different
frictional forces.

6. The Rate of migration of charged particles depends
upon following factors:
– The strength of electric field, shape and size.
– Relative hydrophobicity of the sample.
– Temperature and Ionic strength of the buffer.
– Molecular mass of the taken biomolecule.
– Net charge density of the bio molecule
taken.
– Shape of the taken biomolecule.

7. • V. current:
•
It therefore appears that it is possible to accelerate an electrophoretic
separation by increasing the applied voltage, which ultimately results in
corresponding increase in the current flowing.
• The distance migrated by the ions will be proportional to both current and
time.
• vi. Heat:
• One of the major problems for most forms of electrophoresis, that is the
generation of heat.
• Most of the power generated is dissipated as heat.
• The following effects are seen on heating of the electrophoretic medium
has:
– An increased rate of diffusion of sample and buffer ions which leads to the broadening
of the separated samples.
– Formation of convention currents, which leads to mixing of separated samples.
– Thermal instability of samples that are sensitive to heat.
– A decrease of buffer viscosity and hence reduction in the resistance of the medium.
• If a constant voltage is applied, the current increases during
electrophoresis owing to the decrease in resistance and this rise in current
increases the heat output still further.
• For these reasons, often a stabilized power supply is used, which provides
constant power and thus eliminates fluctuations in heating.

8. • Vii. Elecrtroendosmosis
• This phenomenon is due to the presence of charged groups
on the surface of the support medium.
• For instance, paper has some carboxyl group present,
agarose contains sulfate groups depending on the purity
grade and the surface of glass walls used in capillary
electrophoresis contains silanol (Si-OH) groups.
• These groups, at appropriate pH, will ionize, generating
charged sites.
• It is these charges that generate electroendosmosis.
• In case of capillary electrophoresis, the ionized sianol
groups creates an electrical double layer, or a region of
charge separation, at the capillary wall/electrolytic
interface.
• When voltage is applied cations in the electrolyte near the
capillary walls migrate towards the cathode, pulling
electrolyte solution with them.
• This creates a net electro-osmotic flow towards cathode.

9. Electrophoresis is basically of two
types
• Free boundary or moving boundary
electrophoresis.
• Zone electrophoresis.

10. Moving boundary electrophoresis
• It is a type of electrophoresis without supporting
media, in a free solution.
• Tiselius developed this type of electrophoresis in
1937.
• For the separation of different charged molecules
in a mixture, sample is placed in glass, which is
connected to the electrodes. On applying electric
potential across the tube, charged molecule
migrates towards one or another electrode.

11. Zone electrophoresis
• It involves the separation of charged particles
on inert matrix, or supporting or stabilizing
media.

12. Types of support media used in
electrophoresis:
• Agarose gel
• Agarose- a linear polysaccharide (M.W. 12000 Da) made up of the basic
repeat unit of agarobiose (which comprises alternating units of galactose
and 3,6-anhydrogalactose.
• It is one of the components of agar, that is a mixture of polysaccharides
from seaweeds.
• Agarose gel is formed by suspending dry agarose in aqueous buffer and
then boiling the mixture till it becomes clear solution, which is then
poured and allowed to cool at room temperature to form rigid gel.
• The gelling properties is attributed to inter and intramolecular H-bonding
within and between long agarose chains.
• The pore size of the gel is controlled by the initial concentration of
agarose, large pore size corresponds to low concentration and vice versa.
• Although free from charges, substitution of the alternating sugar residues
with carboxyl, methoxyl, pyruvate, and sulfate groups occur to varying
degrees which can result in electroendosmosis during electrophoresis.

13. • Polyacrylamide gel:
• Cross-linked polysaccharide gel are formed from the polymerization of
acrylamide monomer in the presence of small amount of N,N’-
methylene bis acrylamide (aka- bis-acrylamide).
• Bis-acryl amide is basically two acrylamide molecules linked by a
methylene group, and is used as a cross-linking agent.
• Photopolymerisation is an alternative method that can be used to
polymerize acrylamide gels.
• The acrylamide gel can be made with a content between 3% and 30%
acrylamide.
• Thus, the low percentage gels (e.g., 4%) have large pore size and are
used for electrophoresis of protein- example flat bed isoelectric
focusing, or stacking gel system of an SDS-PAGE.
• Low percentage acrylamide gels are also used to separate DNA.
• Gels between 10% and 20% acrylamide are used in techniques such as
SDS-gel electrophoresis, where smaller pore size now introduces a
sieving effect that contributes to the separation of proteins according
to their size.

14. Instrumentation of electrophoresis:
• Equipment required for electrophoresis basically consists of
two items, a power pack and an electrophoresis unit.
• Electrophoresis units are available for running either
vertical or horizontal gel systems.
• Vertical slab gel units are commercially available and
routinely used to separate proteins in acrylamide gels.
• The gel is formed between two glass plates, clamped
together but held apart by plastic spacers.
• Gel dimensions are typically 12cmX 14cm, with a thickness
of 0.5 to 1mm.
• A plastic comb is placed in the gel solution and is removed
after polymerization to provide loading wells for samples.
• .

15. • When the apparatus is assembled, the lower electrophoresis, tank
buffer surrounds the gel plates and affords some cooling of the gel
plates.
• The gel is cast on a glass or plastic plates and placed on a cooling
plate (an insulated surface through which cooling water is passed to
conduct away generated heat.)
• Connection between the gel and electrode buffer is made using a
thick wad of wetted filter paper, however the agarose gels for DNA
electrophoresis are run submerged in the buffer.
• The powerpack supplies a direct current between the electrodes in
the electrophoresis unit.
• All electrophoresis is carried out in an appropriate buffer, which is
essential to maintain a constant state of ionization of the molecules
being separated.
• Any variation in pH would alter the overall charge and hence the
mobilities (rate of migration in the applied field) of the molecules
being separated

16. On the basis of supporting media, it is
of following types
• Paper electrophoresis
• Cellulose acetate electrophoresis
• Capillary electrophoresis
• Gel electrophoresis.
The equipment used for the electrophoresis
basically consists of two items:
• An electrophoretic unit and a power pack.

17. Electrophoretic unit is of two types:
• Vertical electrophoretic unit
• Horizontal electrophoretic unit

18. vertical electrophoretic unit
• It is also known as vertical slab gel units.
• It is available commercially and mostly used to
separate proteins in acrylamide gels.
• In this, gel is formed between the two glass
plates that are clamped together but uses
plastic spacers to hold them apart.
• Dimensions of gel are typically: 8.5 cm wide X
5 cm high, Thickness = 0.51 mm

19. Horizontal electrophoretic unit
• In this, a gel is cast on a plate, horizontally and
submerged in a running buffer.
• This apparatus is mostly used to separate
nucleic acid or proteins in agarose gel.

20. Paper electrophoresis

21. • Filter paper is very popular as a stabilizing media and
most commonly used for the study of normal and
normal plasma proteins.
• For electrophoresis Chromatography paper is most
suitable and it needs no preparation other than to be
cut to size.
• Apparatus: It consists basically of two items, a power
pack and an electrophoretic cell.
• The power pack provides a stabilized direct current and
has controls for both voltage and current output.
Power packs, which have an output of 0-500 V and 0-
150 rnA are available and can be programmed to give
either constant voltage or current.

22. Cellulose acetate electrophoresis

23. • Kohn in 1958 introduced, Cellulose acetate as a
medium for electrophoresis.
• It was developed from bacteriological cellulose acetate
membrane filters and is commercially available as high
purity cellulose acetate strips, which are thin and have
a uniform micropore structure.
• Cellulose acetate is especially used for clinical
investigations such as separation of hemoglobin’s from
blood, lipoproteins and glycoproteins.
• Buffers used in both the electrophoresis i.e., in paper
and cellulose acetate electrophoresis are same

24. Capillary electrophoresis

25. • It can be described as the new generation electrophoretic
technique.
Commercially available CE instruments consist of
• An electrolyte-filled capillary, which passes through the
optical center of a detector
• A sample injector
• A high voltage power supply
• an auto-sampler. The entire instrument is computer
controlled.
• This technique of electrophoresis has become very popular
over the years because of the many advantages that it has
over conventional electrophoresis techniques.

26. These advantages are listed below:
• Very high-level automation is possible.
• Fast analysis times.
• Detection of separated peaks is done online; thus,
detection is a process that goes hand in hand with
separation and not post-separation as is the case with
conventional techniques.
• Heat generated inside the capillary is effectively dissipated
through the walls of the capillary; therefore, high voltages
can be applied.
• High voltages mean a rapid separation.
• This faster separation along with online detection makes
this technique considerably faster as compared to the
conventional techniques.

27. Gel electrophoresis

29. • It is simple, rapid and sensitive analytical
technique for the separation of charged particle.
• The gels, however, are porous and the size of the
pores relative to that of the molecule determines
whether the molecule will enter the pore and be
retarded or will bypass it. The separation thus not
only depends on the charge on the molecule but
also on its size.
• That resolution of a sample is sharper and better
in a gel than in any other type of medium.
• Gel electrophoresis uses the gel as supporting
media for separation
of DNA, RNA and protein under the influence of
electric charge.

30. Type of gel electrophoresis
• Agarose gel electrophoresis
• SDS-PAGE
• Pulse field gel electrophoresis (PFGE)
• 2D gel electrophoresis

31. Agarose gel electrophoresis
• The supporting media in this type of electrophoresis is
agarose gel.
• It is routinely used for the electrophoresis of nucleic acid like
DNA and RNA.
• Principle
• When potential difference is applied across the electrodes in a
horizontal electrophoretic tank containing agarose gel and
biomolecules (such as nucleic acid) is loaded, then molecules
separated accordingly by their molecular size and move to
their respective electrodes which is tracked with the help of
dye.
• Chemical component such as:
• Media: agarose gel
• Buffer: TAE/TBE (tris acetate EDTA/ Tris borate EDTA).
• Dye: EDTA

33. SDS-PAGE Electrophoresis
• Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis is
mainly used for the separation of proteins based on their mass.
• Principle: This technique uses anionic detergent sodium dodecyl
sulfate (SDS) which disassociates proteins into their individual
polypeptide subunits and gives a uniform negative charge along each
denatured polypeptide. When these denatured polypeptides are
loaded at the cathode end of an electric field, then we get clear bands
of proteins arranged in decreasing order of their molecular mass from
the cathode to anode.
• To separate proteins it involves the use of vertical gel apparatus.
• Chemical component
• Media: SDS-PAGE
• Buffer: lower reservoir: amine buffer with HCl (running gel), Upper
reservoir: amine buffer with glycine (stack gel)
• Stains or dye: Coomassie brilliant blue R-250 (CBB) – anionic dye.

35. Pulse field gel electrophoresis
• It is using to separate large DNA molecule by applying
gel matrix as electric field that periodically changes
direction.

36. 2D gel electrophoresis
• It is type of gel electrophoresis, which is
generally using to analyze proteins.