Shefta Naz

1. Presentation on
GEL ELECTROPHORESIS
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Presented by-
SHEFTA NAZ
M. Pharm (1st sem)
DEPARTMENT OF PHARMACOLOGY
In Supervision of-
Dr. SAVITA UPADHYAY
Assoc. Prof.
Hygia Institute of Pharmaceutical Education & Research
Lko.

2. CONTENTS
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 Introduction
 Principle
 Types of gels
 Preparation and running of agarose gel
 Sampling
 Instrument & Working
 Applications

3. INTRODUCTION
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Electrophoresis is the movement of macromolecules in a
solution(gel) chiefly proteins and nucleic acids (i.e., DNA
and RNA) and their fragments when an electric field is
applied.
Electrophoresis of anions
(negatively charged
particles) is called
Anaphoresis.
Electrophoresis of cations
(positively charged
particles) is called
Cataphoresis.

4. Gel Electrophoresis
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Principle:
Positive and negative
electrical charges are
frequently associated with
biomolecules. When
placed in an electric field,
charged biomolecules
move towards the
electrode of opposite
charge due to the
phenomenon of
electrostatic attraction. ELECTROPHRETIC CHAMBER

5. Gel Electrophoresis
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 Macromolecules are separated on the basis of their size
and charge.
 DNA and RNA are negatively charged molecules, they
will be pulled toward the positively charged end of the gel.
The molecules travel through the pores in the gel at a
speed that is inversely related to their size.
This means that a small DNA molecule will travel a
greater distance through the gel than will a larger DNA
molecule.

6. Gel Electrophoresis
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7. Types of gels:
 Agarose gel:
For separating larger nucleic acids.
 Polyacrylamide gel:
For separating smaller nucleic acids.
- SDS-PAGE - for denaturing the proteins.
 Starch:
Non-denatured proteins can be separated according to
charge and size.
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8. Type
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Source Use Concentration
Agarose
Polysaccharide
polymer extracted
from seaweed
Separate the proteins
ranging in size from
200 kDa
0.7 % - large DNA
2 % - small
fragments
Polyacrylamide
Starch
Chemical Separating proteins 6 %, 8 %, 10 %,
polymerization ranging in size from 5 12 %
reaction
Potato starch
to 2,000 kDa
proteins can be
separated 5-10%

9. • Buffers in gel electrophoresis are used to provide ions
that carry a current and to maintain the pH at a relatively
constant value.
• Tris/Acetate/EDTA (TAE) – pH value -8.0
(tris- “trisaminomethane”)
• Tris/Borate/EDTA(TBE) – pH value -8.0
Buffers :
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10. Preparation of Agarose gel
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• Measure 1 gm of Agarose powder.
• Mix it with 100 ml 1x TAE in a microwavable flask.
• Microwave for 1-3 min until the agarose is completely
dissolved .
• Do not overboil the solution, as some of the buffer will
evaporate and thus alter the final percentage of agarose
in the gel.

11. 11
1. Sampling
2. Electrophoretic run
3. Staining
4. Detection & Quantification
Instrumentation:

12. Sampling:
• The sample to analyse is
optionally mixed with a
chemical denaturant if so
desired, usually SDS for
proteins or urea for nucleic
acids.
• Then the samples are allow to fall in to the sample wells.
• The sample may applied as a spot about 0.5cm in
diameter or as uniform streak.
• The sample is then placed into the electrophoretic
chamber in contact with the buffer.
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13. Electrophoretic Run
•An electric field is applied across the gel, causing the
negatively charged proteins or nucleic acids to migrate across
the gel away from the negative electrode.
•Smaller biomolecules travel farther down the gel, while
larger ones remain closer to the point of origin.
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14. Instrumentation
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15. Visualization:
• DNA may be visualized using ethidium
bromide which when intercalated into DNA,
gives fluorescence under ultraviolet light,
while protein may be visualized using silver
stain or Coomassie Brilliant Blue dye.
• The gel tray is carefully removed and gel from
the electrophoretic chamber and placed onto
the UV light box of the photo documentation
system.
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PAGE of rotavirus proteins stained with
Coomassie blue

16. Applications:
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• Gel electrophoresis is used in forensics, molecular
biology, genetics, microbiology and biochemistry.
• Estimation of the size of DNA molecules following
restriction enzyme digestion (to prepare DNA for
analysis) , e.g. in restriction mapping of cloned DNA.
• Analysis of lipo proteins.
• Used in DNAfingerprinting.

17. • Separation of organic acids, alkaloids, carbohydrates,
amino acids, nucleic acids.
• Used for DNA sequencing.
• Separation of serum proteins.
• Also used in antigen-antibody studies.
• Used in food industry.
• Used to study the protein mixtures.
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18. References:
•Instrumental methods of chemical analysis by B. K. Sharma;
chapter-13 ( Pg. no. C-268, 269, C-280,281 ).
•Instrumental analysis by skoog, Holler, Crouch; chapter-30
( Pg. no. 946, 947 ).
•Skoog & west fundamentals of analytical chemistry by F.
James Holler Stanley R. Crouch; chapter-34 ( Pg. no. 942 ).
•Gel electrophoresis & its applications by Pulimamidi
rabindra reddy and Nomula Raju.
• Poly acryl amide gel electrophoresis from Wikipedia, the
free encyclopedia. 19

19. THANK YOU
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