Electrophoresis
Definition- The term electrophoresis describes the migration of a charged particles
under the influence of an electric field. Various essential biological molecules, such as
amino acids, peptides, proteins, nucleic acids, nucleotides, have ionizable group, which
at given pH exist in a solution as electrically charged species either as cation (+ve) and
anion (-ve) are separated by electrophoresis Under the influence of electric field these
charged particles will migrates either to cathode or anode depending on the nature of
their net charge
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 samples reaches the electrode but the
molecules will have been already separated according to their electrophoretic mobilities.
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.
iii. Frictional force: 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.
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.
V. current: Ohm’s law: V/I=R. 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.
vi. Heat: One of the major problems for most forms of electrophoresis, that is the
generation of heat.
During electrophoresis, the power (W) generated in one supporting medium is given
by W= I2R. 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.
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.
It is used at a concentration between 1% and 3%.
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.
These gels are used for the electrophoresis of both proteins and nucleic acids.
For proteins, the pore size of a 1% agarose gel is large relative to the sizes of
proteins.
, Therefore, used in techniques such as immune-electrophoresis or flat-bed isoelectric
focusing, where proteins are required to move unhindered in the gel matrix to their
native charge.
Such large pure gels are also used to separate much larger molecules such as RNA
and DNA, because the pore sizes are still large enough for RNA and DNA molecule
to pass through gel.
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.
Acrylamide monomers is polymerized in head to tail fashion into long chain, thus
introducing a second site for chain extension.
Proceeding in this way, a cross-linked matrix of fairly well-defined structure is
formed.
The polymerization of acrylamide is an example of free radical catalysis and is
initiated by the addition of ammonium persulfate and the base N, N, N’, N’- tetra-
methylene diamine (TEMED).
TEMED catalyses decomposition of the persulphate ion to give free radical.
a) S2O82- + e– à SO42- + SO-•4
b) R• + M à RM•
c) RM•+ M à RMM•
d) RMM•+M à RMMM• and so on…
Photopolymerisation is an alternative method that can be used to polymerize
acrylamide gels.
Photodecomposition of riboflavin generates a free radical that initiates
polymerization.
Acrylamide gels are defined in terms of the total percentage of acrylamide present,
and the pore size in the gel can be varied by changing the concentration of
acrylamide and bis-acrylamide.
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.
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.
A plastic comb is placed in the gel solution and is removed after polymerization to
provide loading wells for samples.
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.
Definition- The term electrophoresis describes the migration of a charged particles
under the influence of an electric field. Various essential biological molecules, such as
amino acids, peptides, proteins, nucleic acids, nucleotides, have ionizable group, which
at given pH exist in a solution as electrically charged species either as cation (+ve) and
anion (-ve) are separated by electrophoresis Under the influence of electric field these
charged particles will migrates either to cathode or anode depending on the nature of
their net charge
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 samples reaches the electrode but the
molecules will have been already separated according to their electrophoretic mobilities.
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.
iii. Frictional force: 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.
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.
V. current: Ohm’s law: V/I=R. 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.
vi. Heat: One of the major problems for most forms of electrophoresis, that is the
generation of heat.
During electrophoresis, the power (W) generated in one supporting medium is given
by W= I2R. 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.
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.
It is used at a concentration between 1% and 3%.
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.
These gels are used for the electrophoresis of both proteins and nucleic acids.
For proteins, the pore size of a 1% agarose gel is large relative to the sizes of
proteins.
, Therefore, used in techniques such as immune-electrophoresis or flat-bed isoelectric
focusing, where proteins are required to move unhindered in the gel matrix to their
native charge.
Such large pure gels are also used to separate much larger molecules such as RNA
and DNA, because the pore sizes are still large enough for RNA and DNA molecule
to pass through gel.
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.
Acrylamide monomers is polymerized in head to tail fashion into long chain, thus
introducing a second site for chain extension.
Proceeding in this way, a cross-linked matrix of fairly well-defined structure is
formed.
The polymerization of acrylamide is an example of free radical catalysis and is
initiated by the addition of ammonium persulfate and the base N, N, N’, N’- tetra-
methylene diamine (TEMED).
TEMED catalyses decomposition of the persulphate ion to give free radical.
a) S2O82- + e– à SO42- + SO-•4
b) R• + M à RM•
c) RM•+ M à RMM•
d) RMM•+M à RMMM• and so on…
Photopolymerisation is an alternative method that can be used to polymerize
acrylamide gels.
Photodecomposition of riboflavin generates a free radical that initiates
polymerization.
Acrylamide gels are defined in terms of the total percentage of acrylamide present,
and the pore size in the gel can be varied by changing the concentration of
acrylamide and bis-acrylamide.
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.
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.
A plastic comb is placed in the gel solution and is removed after polymerization to
provide loading wells for samples.
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.
