electrophoresis presentation

1. ELECTROPHORESIS
MADE BY:- AMAN SHARMA
SUBJECT:- BIOPHYSICS
LECTURER:- OLIA RCHEULISHVILI

2. INTRODUCTION
 Electrophoresis is the motion of dispersed
particles relative to a fluid under the influence of a
spatially uniform electric field. Electrophoresis of
positively charged particles (cations) is sometimes
called cataphoresis, while electrophoresis of negatively
charged particles (anions) is sometimes
called anaphoresis.

3. INTRODUCTION
(CONT.)
 Electrophoresis is a laboratory technique used to
separate DNA, RNA, or protein molecules based on
their size and electrical charge. An electric current is
used to move molecules to be separated through a gel.
Pores in the gel work like a sieve, allowing smaller
molecules to move faster than larger molecules. The
conditions used during electrophoresis can be adjusted
to separate molecules in a desired size range.

4. FACTORS
AFFECTINGRATE
OFMIGRATION
OFCHARGED
PARTICLES
 The rate of migration of charged molecules depends
upon the following factors:
1. The strength of electric field.
2. Relative hydrophobicity of the sample.
3. Ionic strength and temperature of the buffer.
4. Molecular size of the biomolecule.
5. Net charge density of the biomolecule.
6. Shape of the biomolecule.

5. ELECTROPHORETIC
MOBILITY
 Electrophoretic mobility is defined as the rate of
migration (cm/s) per unit field strength (volts/cm).
 Where,
 μ= electrophoretic mobility
 Q= net charge of the ion
 r= ionic radius of solute
 η= viscosity of medium

6. ELECTROPHORESIS
APPARATUS

7. ELECTROPHORESIS
APPARATUS
(CONT.)
1. Buffer Tank – to hold the buffer
2. Buffer
3. Electrodes – made of platinum or carbon
4. Power supply
5. Support media
 NOTE: choice of buffer depends on the nature of the
substance to be separated and the electricity is
supplied at a constant current and voltage.

8. SUPPORT
MEDIA
1. PAPER:
A. filter paper such as Whitman no. 1 and no. 3 MM.
2. CELLULOSE ACETATE:
A. containing 2 to 3 acetyl groups.
B. to give sharper bands.
3. GELS:
A. 3D semisolid colloids.
B. enhanced resolving power
C. prepared from starch, agar and polyacrylamide.

9. TYPES
ELECTROPHORESIS
FRONTAL ZONAL
PAPERMICRO MOVING CELLULOSE
ACTATE
GEL

10. FRONTAL
ELECTROPHORESIS
 In this type of electrophoresis a free electrolyte is
taken in place of supporting media.
 It is of two types:
A. micro electrophoresis which is used in calculation of
zeta potentials.
B. moving boundary electrophoresis which is used for
quantitative analysis of complex mixtures of
macromolecules, especially proteins.

11. ZONAL
ELECTROPHORESIS
 It is the most prevalent electrophoretic technique these
days.
 The process is carried out on a supporting media.
 It is of three types out of which gel electrophoresis is
used most widely.
 It produces zones of proteins that are heterogeneous an
physically separated from each other.
 It is classified according to structure of the supporting
material, for e.g. AGE, CAE and PAGE etc.

12. A simplified schematic drawing of a protein pattern
from the serum of a subject with haptoglobin type 2-
1 (separation by PAGE). Some zones contain more
than the one protein shown, as demonstrated by
immunological techniques. AAT, Alpha; antitrypsin;
ALB, albumin; AMG, alpha-macroglobulin; BLP,
beta-lipoprotein; C3, complement 3; FIB, fibrinogen;
gamma, gamma-globulin; HP, haptoglobin; TRF,
transferrin.

13. GEL
ELECTROPHORESIS
 Gel electrophoresis involves the use of gel as
supporting media for separation of DNA, RNA or
proteins under the influence of electric charge.
 It is usually performed for analytical purposes but may
be used as a preparative technique to partially purify
molecules prior to use for other methods such as mass
spectrometry, PCR, cloning, DNA sequencing and
immune - blotting.
 This is the most commonly used electrophoresis in
biotechnology laboratories and is used for almost all
types of experiments in RD.
 Ethidium bromide is used for staining in the process.

14. PRINCIPLE
 When a potential difference is applied across the
electrodes of a horizontal electrophoretic tank containing
agarose gel and biomolecules (such as nucleic acids) are
loaded, then they get separated according to their
molecular size (bigger molecules have more molecular size
and smaller molecules have small molecular size) and
move to their respective electrodes. Here the agarose gel
acts as a sieve.
 As in a sieve the large particles stay above and the
particles which are smaller than the pore size passes
through it, similarly in the gel the larger and the bulky
molecules stay behind whereas the smaller molecules
move faster and quickly towards their respective
electrodes.
 This process may be imagined like a running competition.
The one who is thinner and have a flexible body will be at
the ending point sooner than the one who is fat and bulky.

15. APPLICATIONS
 Separation of restriction enzyme digested DNA
including genomic DNA, prior to Southern Blot
transfer. It is often used for separating RNA prior to
Northern transfer.
 Analysis of PCR products after polymerase chain
reaction to assess for target DNA amplification.
 Allowing estimation of the size of DNA molecules using
a DNA marker or ladder which contains DNA
fragments of various known size.
 Allows the rough estimation of DNA quantity and
quality.

16. APPLICATIONS
 Quantity is assessed using lambda DNA ladder which
contains specific amounts of DNA in different bands.
 Quality of DNA is assessed by observing the absence of
streaking or fragments (or contaminating DNA bands).
 Other techniques rely on agarose gel electrophoresis
for DNA separation including DNA fingerprinting.

18. ADVANTAGES&
DISADVANTAGES
OFAGAROSEGEL
ELECTROPHORESIS
1. ADVANTAGE:
The gel is easily poured and does not denature the
samples. The samples can also be recovered.
2. DISADVANTAGE:
The gel can melt during electrophoresis and the buffer
may get exhausted due to which different forms of
genetic material may run in unpredictable forms.

19. REFERENCES
 www.Wikipedia.com
 www.genome.gov.com
 www.sciencedirect.com