In this slide contains introduction, principle, methods, factors, application and disadvantage of Horizontal Electrophoresis. Presented by: A.Geethanjali (Department of pharmacology), RIPER, anantapur.

1. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1
A Seminar as a part of curricular requirement
for I year M. Pharm I semester
Presented by
A.Geetanjali
(Reg. No. 20L81S0106)
Dept. Of Pharmacology
Under the guidance/Mentorship of
Mr. A.Sudheer ,M.Pharm
Associate Professor
Dept. of Pharmacology
Horizontal electrophoresis

2. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 2
Introduction
Principle
Materials required for run a gel
Method for electrophoresis
Factors affecting electrophoresis
Applications
Disadvantages
References
Contents

3. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 3
• Electrophoresis is a process of migration of charged particle through
a solution under the influence of external electric field.
• The term electrophoresis was coined from a Greek word “phoresis”
which means “Being Carried Away”.
• The word electrophoresis means “to carry with electricity”.
• Shorter molecules move faster and migrate faster than longer ones.
• Electrophoresis is used extensively in DNA, RNA and protein
analysis.
Introduction

4. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 4
• Separate molecules from each other on the basis of
– size and/or
– charge and/or
– shape
• Basis of separation depends on how the sample and gel are
prepared.
Principle

5. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5
Charge
Separation
Size
Separation
Negative
Molecules
PositiveMolecules
Mixtureof
Charged
Molecules

6. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 6
 Electrophoresis apparatus
 Agarose gel
 Gel casting tray
 Buffer
 Staining agent(dye)
 Comb
 DNA ladder
 Sample to be seperate
Materials required for run a gel

7. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 7
Electrophoresis apparatus

8. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 8
Agarose gel
• Agar is a mixture of polysaccharide extracted from sea weeds.
• Agarose is a highly purified uncharged polysaccharide derived from
agar.
• Agarose is Non-toxic.
• Weigh out the appropriate mass
of agarose into an Erlenmeyer flask.
Agarose gels are prepared using a
w/v percentage solution.

9. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 9
• Melt the agarose.it is done by heating in a
microwave/ Bunsen flame. after that
agarose will be completely dissolved.
• The pores of an agarose gel are large,
agarose is used to separate macro
Molecule such as nucleic acids, large
protein complex
• Add ethidium bromide (EtBr) to a
concentration of 0.5 μg/ml. Alternatively
Note: EtBr is carcinogen
% Agarose
(W/V)
Size range
(Kb pairs)for
optimals
separation
0.5 2-25
0.75 0.7-10
1.0 0.5-10
1.5 0.2-3
2.0 0.1-2
3.0 0.07-1.5
4.0 0.04-0.9
5.0 0.03-0.6
6.0 0.01-0.4

10. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 10
 Gel casting tray
• Available in a variety of sizes and composed of UV-transparent
plastic.
• The open ends of the trays are closed with tape while the gel is
being cast, then removed prior to electrophoresis
• Pour in the melted agarose and insert the gel comb, making sure
that no bubbles are trapped underneath the combs and all bubbles
on the surface of the agarose are removed before the gel sets.

11. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 11

12. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 12
Buffer

13. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 13
Staining agent
• Ethidium bromide the standard concentration used in staining DNA
in gels is 0.5-1ug/mL
• Ethidium bromide is a fluorescent dye that intercalates between
bases of nucleic acids and allows very
convenient detection of
DNA fragments in gels.

14. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 14
• EtBr is having carcinogenic activity.
• Protect from sunlight.
Preparation of Ethidium bromide stock solution :
• Weigh 10mg ethidium bromide into a sterile tube and dissolve in
10ml of sterile distilled water.
• Other alternatives for ethidium bromide :
 Methylene blue
 Syber safe
 xylene cyanol
 bromphenol blue

15. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 15
 Comb :
• The white/red plastic electrophoresis comb has many tines.
• Electrophoresis combs are
used to create the wells in
gels for electrophoresis,
It is often used to analyze
DNA fragments. When a gel
is poured, a comb is inserted.

16. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 16
DNA ladder:
• It is a solution of DNA molecules of
different length.
• DNA Ladder consists of known
DNA sizes used to determine the size
of an unknown DNA sample.
• The DNA ladder usually contains
regularly spaced sized samples which
when run on an agarose gel looks like a
“Ladder".

17. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 17
Voltage:
• The speed of movement through the gel is determined by the voltage
gradient, i.e. the voltage between the electrodes.
•  voltage, rate ofmigration
• Horizontal gel tanks are generally run at between 5 – 10 V / cm so if
your tank has an electrode distance of 10 cm, you would run the gel
at 50 – 100V.
• An ampholyte become positively charged in acidic condition and
migrate to cathode,in alkaline condition they become negatively
charge and migrate to anode

18. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 18
Sample to be separate:
• DNA samples should be prepared in a volume that will not overflow
the gel wells.
• Samples are typically loaded into the wells with a micropipette. Care
should be taken to prevent mixing of the samples between wells.

19. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 19
Method for electrophoresis

20. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 20

21. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 21
The rate of migration of solute in an electric field depends on the
following factors:
• Net charge on the particle
• Mass and shape of the particles
• pH of the medium
• strength of electric field
• Properties of supporting medium
• Temperature
Factors affecting electrophoresis

22. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 22
• DNA sequencing
• For the estimation of size of DNA molecule.
• Agarose gels allow purification of DNA fragments
• Protein research /purification(separate different types of protein
mixtures as well as nucleic acids).
• It is employed in biochemical and clinical fields i.e, in study of
protein mixture such as blood serum, haemoglobins and in the study
of antigen-antibody interactions.
Applications

23. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 23
• Electro osmosis is high.
• Resolution is less compared to polyacrylamide gels.
• Different sources and batchs of agar tend to give different results and
purification is often necessary.
Disadvantages

24. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 24
• Pei Yun Lee,1 John costumbrado. Agarose Gel Electrophoresis for the
Separation of DNA Fragments. Journal of visualized
experiments. 2012; 6(2): 2-5.
• Jialiang Li, Yushi Yang, Zhou Mao. Enhanced Resolution of DNA
Separation Using Agarose Gel Electrophoresis Doped with Graphene
Oxide.Springers.2016; 404: 1-5.
• Smith, S.B., Aldridge, P.K., & Callis, J.B. Observation of individual
DNA molecules undergoing gel electrophoresis. PubMed. 1989; 243:
203-206.
• Voytas.D. Agarose Gel Electrophoresis. Current Protocols in
Molecular Biology. 2001; 23(9): 1-8.
References

25. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 25