Gel electrophoresis is a technique used to separate macromolecules like DNA, RNA, and proteins based on their size and charge. It works by applying an electric current that causes charged molecules to migrate through a gel at different rates depending on factors like their mass and shape. There are different types of gels used like agarose, starch, and polyacrylamide gels. The document discusses the principles, instrumentation, buffers, support media, and applications of gel electrophoresis like determining molecular weights and purifying proteins.

1. Gel Electrophoresis
Submitted To-
Department of Zoology
Isabella Thoburn College
Lucknow.

2. Definition of Electrophoresis
Electro- “Charged particle” ; Phoresis means - “ Being Carried Away”
• The charged particles moves through a media in the
presence of an electric field at a given pH
• Electrophoresis is a separation technique that is based on the
mobility of ions in an electric field
• This technique is used for both DNA and RNA analysis.

3. 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 current.”
The electric force ( Eq) is equal to the friction force or viscous drag ( Fv)
Eq= Fv
Where,
E = Strength of electric field
q = net charge of particle
F= Frictional coffecient
V = Velocity

4. Ohm’s law states that voltage and current are related
by following relationship-
V= iR
Where,
V = Voltage
I= Current
R=
Resistance
• The electric strength (E) is kept constant . Then V ( Velocity) of particles depend on its
net charge (q) and its frictional coffecient(f).
• The frictional coffecient is directly proportional to the radius of a spherical particle . It
can be represented by following equation- V= Eq/ F
Where,
V= Velocity of migration of the molecule
E= Electric field in volts / cm
q = Net charge on the molecule
F= Frictional coffecient

5. Electrophoresis Instrumentation
• A wide range of electrophoretic equipment is available .
• Each has common basic design consists of two parts-
1. Electrophoretic Tank 2. Power supply
Electrophoretic Unit consists of Electrophoresis Media
Electrophoretic Chamber
Buffers
Support Medi
Filter paper Gel

6. Bioinstrumentation By-L. Veera K

7. Buffers
Buffers in gel electrophoresis are used to provide ions that carry a current and to
maintain pH at a relatively constant value.
• Serum protein separation
• Poor resolution , weak buffer
Barbitone Buffer( 8.0 pH)
Phosphate Buffer (pH- 7.5) • Enzyme separation
• Low buffering capacity
:-High conductivity.
Buffers Function
Tris – borate – EDTA buffer
(TBE) pH- 8.0
• Nucleic acid separation
• Good resolution , high buffering
capacity ,low conductivity.
•Nucleic acid separation
• Good resolution , high
buffering capacity ,low
conductivity
Tris – acetate –EDTA buffer
(TAE) pH – 8.0
Tris – glycine buffer (pH-
8.0)
• Protein separation
• High buffering capacity,
low conductivity

8. Support Media
Filter Paper
Gel
Cellulose acetate membrane
• (CAM) is a supporting medium .
• It contained more regularly
defined pore structure separation
could be make in 1- 2 hours.
Agaros
e
Gel
Polyacryl
a-mide
Gel
Starch
Gel
Have
lower
resolving
power for
DNA .
It is
usually
used for
proteins,
and have
very high
resolving
power for
small
fragment
s of DNA.
Partially
hydrolysed
potato
starch
makes for
another non-
toxic
medium for
protein
eectrophore
sis.

9. Types of
Electrophoresis
Free Electrophporesis Zone Electrophoresis
Micro
electrophoresis
Moving
boundary
Paper
Electrophoresis
Cellulose
acetate
electrophoresis
Gel
Electrophoresis

10. Gel Electrophoresis
• Gel electrophoresis is a
method for separation
and analysis of
macromolecules ( DNA ,
RNA , Proteins)
• It is of two types-
Column
Electrophore
sis
Slab Gel
Electrophore
sis
Supports for bottom of gel
Line Diagram Of Slab Gel Apparatus
Source-www.google.com

11. Gel Electrophoresis
Agarose Gel
Electrophoresis
Starch Gel
Electrophoresis
Polyacrylamide Gel
Electrophoresis
Gel
Electrophoresis

12. Agarose Gel Electrophoresis
• Agarose Gel Electrophoresis is method of gel electrophoresis .
• It is used in biochemistry , molecular biology , clinical chemistry to
separate a mixed population of DNA or protein in a matrix of agarose.
• Most agarose gels used are between 0.7-2% dissolved in electrophoresis
buffer.
Casting of
Gel
Loading of
Samples
Electrophoresis
Staining and
visualisation
Down stream
procedures
General Procedure -

13. Source- Bioinstrumentation –By- L.Veera K

14. Applications
 Isolate large number of proteins.
 Identify the purity of isolated proteins.
 Determine sequence of DNA , mutation , molecular weight.
 Detect the precursor molecules of tRNA,rRNA,mRNA.
 Study the kinetics of the interconversion of conformation
in many tRNA’s.

15. Starch Gel Electrophoresis
• Smithies (1995) introduced zone electrophoresis
using Starch gel electrophoresis.
• Resolving power of starch gel is very high .
Starch is
hydrolysed in
acetone at 37 ͦ C .
Suspension
neutrilised with
sodium acetate
Wash with
distilled water
and acetone
Hydrolyse
d starch
is heated
Cool in
buffer set
as gel
Sample is
applied to
gel by
soaking
filter paper
Insert it in
the gel.
Appearance of
coloured band
indicates the
presence of active
enzymes .
General Procedure-

16. Drawbacks of Starch Gel
• Unlimited pore size
contamination of starch gel by
microorganisms.
• Starch gel turns opaque
making direct photoelectric
determination impossible

17. Polyacrylamide Gel Electrophoresis
• PAGE describes a technique widely
used in biochemistry , forensics ,
genetics, molecular biology &
biotechnolgy to separate biological
molecules , usually proteins or nucleic
acids , according to their
electrophoretic mobility.
• For proteins , SDS is an ionic
detergent applied to protein sample to
linearize proteins & to import a
negative to linearized proteins.
• The procedure is called SDS-PAGE.
• Electrophoresis in acrylamide gel is
frequently referred to as PAGE.
Bioinstrumentation By-L. Veera Kum

18. General Procedure-
General
Procedure
1. Sample
Preparation
2. Preparing
Acrylamide Gel
3. Electrophoresis
4. Further
Processing

19. Source- www.google.com

20. Components
use in PAGE
Chemical
Buffer
Ammomiu
m per
sulphate
Urea
Acrylamid
e
Bis
acrylamid
e
Sodium
Dodecyl
sulphate

21. Chemicals for processing and visualization
Chemicals
1.Tracking
Dye
2.Loading
aids
3. Commasive
Brilliant Blue
4. Ethidium
Bromide

22. • Separation is based upon the molecular weight
of proteins.
• The most common method for determining
molecular weight of proteins.
• Very useful for checking of purity of protein
sample
SDS
• Separation is based upon charge, size, &
shape of macromolecules.
• Useful for separation and / or purification of
mixture proteins.
• This was the origin mode of electrophoresis.
Native
PAGE
Types of PAGE

23. Applications
Applications
1. Used for
estimation of
molecular
weight of
proteins and
nucleic acids
3. Purification of
isolated proteins
4. Monitoring changes
of protein content in
body fluids
2. Determination of
subunit structure of
proteins