This document discusses different types of electrophoresis techniques. It begins with an introduction to electrophoresis and its history. It then describes various electrophoresis techniques including gel electrophoresis using agarose gel or polyacrylamide gel, capillary electrophoresis, isoelectric focusing, two-dimensional electrophoresis, and immuno electrophoresis. The document explains the basic principles of electrophoresis and how it uses an applied electric field to separate biomolecules like proteins, nucleic acids, and carbohydrates based on their charge and size. It concludes with some applications of electrophoresis techniques.
1. TYPES OF ELECTROPHORESIS
By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )
3. Introduction
• When a potential difference is applied between the two electrodes in a
colloidal solution, It has been observed that the colloidal particles are
carried to either the positive or negative electrode.
• In other words , they behave as if they are electrically charged w.r.t. the
dispersion medium. This phenomenon is known as electrophoresis.
• Many important biological molecules, such as amino acids, peptides,
proteins, nucleotides and nucleic acids, possess ionisable groups and,
therefore, at any given pH, exist in solution as electrically charged
species either as cations or anions.
• Under the influence of an electric field these charged particles will
migrate either to the cathode or to the anode, depending on the nature
of their net charge.
4. History
• 1939- zone electrophoresis developed.
• 1950- agar gel electrophoresis
• 1959- acrylamide gel first used.
• 1971- SDS electrophoresis.
• 1979- agarose gel electrophoresis.
• 1983- capillary electrophoresis.
5. Electrophoresis
• Electrophoresis is defined as the “migration of charged
molecules under the influence of an external electric field”
• Electrophoresis is a technique used for the separation of
biological molecules based on their movement due to the
influence of a direct electric current.
• The technique was pioneered in 1937 by the Swedish chemist
Arne Tiselius for the separation of proteins.
• It has now been extended to the separation of many other
different classes of biomolecules including nucleic acids,
carbohydrates and amino acids.
6. PRINCIPLE
Any charged ion or molecule migrates when
placed in an electric field. The rate of migration
depend upon its net charge, size, shape and the
applied electric current.
Can be represented by following eq.
E * q
v = ---------------
f
7. • v = velocity of migration of the molecule.
• E = electric field in volts per cm
• q = net electric charge on the molecule
• f = frictional coefficient
The movement of charged particle in an electric field is
expressed in terms of electrophoretic mobility ,denoted by
µ.
where,
µ = v/E OR
µ = q/f
9. GEL ELECTROPHORESIS
• It is a technique used for the separation of
Deoxyribonucleic acid, Ribonucleic acid or
protein molecules according to their size and
electrical charge using an electric current applied
to a gel matrix.
• Types of Gel:
Agarose gel.
Polyacrylamide gel.
11. Agarose Gel Electrophoresis
A highly purified uncharged polysaccharide
derived from agar.
Used to separate macromolecules such as nucleic
acids, large proteins and protein complexes.
The cross linked structure gives the gel good
anticonventional properties.
12. POLYACRYLAMIDE GEL
ELECTROPHORESIS
Commonly used components: Acrylamide monomers,
Ammonium persulphate, Tetramethylenediamine
(TEMED), N,N’-methylenebisacrylamide.
These free radicals activate acrylamide monomers
inducing them to react with other acrylamide
monomers forming long chains.
Used to separate most proteins and small
oligonucleotides because of the presence of small
pores.
13. Isoelectric focusing
When electrophoresis is run in a solution buffered at
constant pH , proteins having a net charge will migrate
towards the opposite electrode so long as the current flows
Electrophoretic method that separates proteins according
to the iso-electric points
Seperation is achieved by applying a potential difference
across a gel that contain a pH gradient
Isoelectric focusing requires solid support such as agarose
gel and polyacrylamide gel
14.
15. Capillary Electrophoresis
• Capillary electrophoresis, is the technique of performing
electrophoresis in buffer-filled, narrow-bore capillaries,
normally from 25 to 100 pm in internal diameter .
• The capillary is filled with electrolyte solution which conducts
current through the inside of the capillary. The ends of the
capillary are dipped into reservoirs filled with the electrolyte.
• Electrodes (platinum) are inserted into the electrolyte reservoirs
to complete the electrical circuit
16.
17. 2D- Electrophoresis
• Two-dimensional gel electrophoresis is
widely used to separate complex mixtures of
proteins into many more components than is
possible in conventional one-dimensional
electrophoresis.
• Each dimension separates proteins
according to different properties.
18. Immuno Electrophoresis
It is a biochemical methods for separation and
characterization of proteins based on electrophoresis and
reaction with antibodies . All variants of
immunoelectrophoresis require immunoglobulin, also
known as antibodies reacting with the proteins to be
separated or characterized.
The agarose was chosen as the gel matrix because it has
large pores allowing free passage and separation of
proteins, but provides an anchor for the immuno
precipitates of protein and specific antibodies
19. Application
• Determination of gene sequence
• Isolation of entire chromosome.
• separation and characterization of protein.
• DNA fingerprinting evidence.
• Restriction mapping of DNA