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Electrophoresis -Principles and Methods
Presentation · May 2020
DOI: 10.13140/RG.2.2.22115.99368
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 Indian Council of Agricultural Research

Lecture-1
Electrophoresis – Principles and Methods

Dr. Ranjeet R. Kumar

Sr. Scientist

5/29/2020 E-mail:ranjeetranjaniari@gmail.com
 Outline of Talk

• Introduction
• Basic principle
• Types
• Brief description about each technique [why, how, where,
outcome]
• Examples

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 Arne Wilhelm Kaurin Tiselius (10 August 1902 – 29
October 1971)

• Swedish biochemist
• Won the Nobel Prize in Chemistry in 1948
• Research on electrophoresis and adsorption analysis
• Discovered the complex nature of the serum proteins.

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 Introduction

Electrophoresis - the migration and separation of charged

particles (ions) under the influence of an electric field.

It is the movement of charged particles through an electrolyte

when subjected to an electric field at given pH.

Electrophoresis

Electro Phoresis

Electron/ Separation
Current
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 Related Terms

• Anaphoresis - Electrophoresis of anions or negatively charged particles.

• Cataphoresis - Electrophoresis of cations or positively charged particles.

• Iontophoresis – Migration of small ions.

• Zone Electrophoresis - migration of charged macromolecules in porous

support medium such as paper, cellulose acetate or agarose gel film.

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 Electrophoretic Mobility = V/E = q/f

Frictional Resistance (f) α -

Hydrodynamic size of the molecule
Shape of the molecule
Pore size of the medium
Viscosity of buffer

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 Rate of Migration

Directly proportional to Inversely proportional to

• Current • Shape
• Charge • Size
• Voltage • Frictional resistance

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 Charge

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 Sieving effect and
shape and size of
molecules
• Size – bigger the molecule,
greater the frictional and
electrostatic forces exerted by the
medium
• Larger particle ahs smaller µ
• Smaller particle has larger µ

• Shape – Round exert lesser

frictional and electrophoretic
retardation

• Globular protein moves faster

than the fibrous protein.

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 Common Buffer Used

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 Supporting Medium

It is an matrix in which the molecules (DNA/RNA/ Proteins) separation takes place

Supporting Medium

Paper
Cellulose acetate
Agar gel
Starch grains
Polyacrylamide gel
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 Agarose Gel

• Three-dimensional matrix formed of

helical agarose molecules.

• Supercoiled bundles that are aggregated into three-

dimensional structures.

• Has channels and pores through which

biomolecules can pass.
• Agarose is a polysaccharide.

• Extracted from red seaweed.

• It is a linear polymer made up of the

repeating unit of agarobiose.

• Disaccharide made up of D-galactose

and 3,6-anhydro-L-galactopyranose.
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 Agarose Gel Structure

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 Polyacrylamide Gel

• Polyacrylamide gels are created by the

polymerization of acrylamide monomers with the
N,N-methlylenebisacrylamide cross-linker.
• The pore size, formed within the gel, is dependent
on the amount of cross-linking and the lengths of
the polymer chains.
• Ammonium persulphate is usually used as the
free radical initiator
• N,N,N',N'-tetramethylenediamine (TEMED)
stabilizes the polymerization chain reaction.
• The chain reaction is inhibited by molecular
oxygen
• Polymerization is conventionally carried out
between two thin glass plates with the top of the
gel solution covered with water-saturated butanol.

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 Difference between Agarose and Polyacrylamide Gel

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 Electrophoretic Unit

Horizontal Vertical

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 NEXT LECTURE

• Types of Electrophoresis

• Brief Description about each technique

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View publication stats

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