Electrophoresis is a process that separates charged biomolecules based on their movement in an electric field, influenced by factors such as voltage, frictional resistance, and the properties of the medium used. The method can utilize various support media, including agarose and polyacrylamide gels, each affecting the separation efficiency and quality. Heat generated during electrophoresis can impact sample integrity, leading to issues like diffusion and denaturation.

1. Electrophoresis
Amandeep Singh
Assistant Professor
Department of Biotechnology
GSSDGS Khalsa College Patiala

2. Electrophoresis
Migration of charged particles under the influence of electric field.
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3. Biomolecules have ionizable groups, at any given
pH, exist in solution as electrically charged species
(Cations or Anions)
Cations
Cathode
Anion
Anode
Under influence
of electric field
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4. Electrophoretic Equipment
Electrophoretic Unit Power Supply
Horizontal Vertical
Agarose Gel Electrophoresis SDS-PAGE
Supply DC between
electrodes in
electrophoretic unit
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5. How Charged particles separate?
• When potential difference (voltage) is applied across the electrode, it
generates potential gradient (E).
E = V (applied voltage)
d (distance between electrodes)
• When this potential gradient is applied, force on molecule bearing
charge of q coulombs is Eq newtons.
• It is this force that drives a charged molecule toward the electrode.
• There is also a frictional resistance that retards the movement of this
charged molecule.
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6. Voltage
V
Potential gradient
E
Force = Eq newton
Charge q
Electrode
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7. Frictional resistance depends on
• Size of molecule
• Shape of molecule
• Pore size of medium
• Viscosity of buffer
Velocity of an charged molecule in an electric field is given by
V = Eq
f
f= frictional coefficient
Increase in voltage lead to increase in velocity, but increase in voltage also
lead to generation of heat, which is major problem in electrophoresis.
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8. Effect of heating
• Increase rate of diffusion of sample and buffer ion that results in
broadening of separated sample
• Formation of Convection currents: leads to mixing of separated
sample
• Heat sensitive samples become denature
• Decrease in buffer viscosity leads to reduction in resistance of the
medium
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9. Low voltage
Low Current
Poor separation
High voltage
High Current
High Heating
Constant voltage
High Current
High Heating
Solution
Stabilizer
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10. Support Media
Agarose
• It is a linear polysaccharide made up of basic unit of agarobiose.
• Agarobiose is alternating units of galactose and 3,6-unhydrogalactose.
• Pore size: Large pore size results from small concentration of agarose
Small pore size results from high concentration of agarose
Polyacrylamide gel
• Electrophoresis in acrylamide is called Poly-Acrylamide Gel
Electrophoresis (PAGE).
• Polyacrylamide gel is made by bisacrylamide and acrylamide joined
together by methylene group.
• Polymerization of acrylamide is the example of free radical catalysis
initiated by addition of ammonium per sulphate (APS) and N,N,N,N- tetra
methylene diamine (TEMED).
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