Paper and gel electrophoresis.pdf

It's my prepared presentation on paper and gel electrophoresis for m.pharm students of 1st year pharmaceutics department.
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1. PAPER ELECTROPHORESIS AND GEL
ELECTROPHORESIS
Guide by : Dr. Vijay Borkar
Presented by: Priya Talekar
Date: 12/02/2022
RAJARSHI SHAHU COLLEGE OF PHARMACY
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2. CONTENTS
•Theory
•Principal
•Instrumentation
•Factors affecting
•Paper Electrophoresis
•Gel electrophoresis
•Application
•Reference
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3. THEORY OF ELECTROPHORESIS
•Electrophoresis is a chromatography technique used to separate mixture
of charged molecules in a fluid or gel based on their charge, binding
affinity, and size under an electric field.
•In the year 1807, Ferdinand Frederic Reuss was the first person to
observe electrophoresis. He was from Moscow State University.
Anaphoresis is the electrophoresis of negative charge particles i.e.anions
whereas cataphoresis is electrophoresis of positive charge ions i.e.
cations.
•Electrophoresis has a wide application in separating and analysing
biomolecules like proteins, plasmids, RNA, DNA, nucleic acids.
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4. 4

5. PRINCIPAL
•Electrophoresis system consists of two electrodes of opposite charged
(anode and cathode), connected by a conducting medium called an
electrolyte.
•Ions that are suspended between two electrodes tends to travel towards
the electrodes that bears opposite charges.
•The Separation effect on the ionic particles results from differences in
their velocity(v) which is product of particles mobility(m) and the field
strength(E)
•i.e.V=mE
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6. FACTORS AFFECTING
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7. TYPES OF ELECTROPHORESIS
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8. ZONE ELECTROPHORESIS
•Involves the migration of the charged particle on the supporting media can
be Paper, Gel,etc.
•In this type of electrophoresis the separation process is carried out on a
stabilizing media
•ADVANTAGES:
•Useful in biochemical investigations.
•Small quantity of sample can be analysed.
•Cost is low and easy maintenance.
•DISADVANTAGES:
•Unsuitable for accurate mobility and isoelectric point determination. Due
to the presence of supporting medium, technical complications.8

9. PAPER ELECTROPHORESIS
•It is a technique that use whatman filter paper number 1,
which is moisten by a buffer and then connected by two
ends electrode.
•The samples are spotted in the center of the paper, voltage
is applied, and the spots migrate according to their charges.
•After electrophoresis, the separated components can be
detected by a variety of staining techniques, depending
upon their chemical identity.
•Staining technique like bromophenol blue staining,zinc
staining,silver staining, fluorescent staining,etc
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10. •Paper:
•Used as the supporting medium. Normally Whatman filter paperof suitable
dimension with a length so that both end of the strip of paper touch the buffer
solution, kept in the electrolyde vessels.
•Electrodes and voltageto be applied:
•The electrode in the form of a thin wire is made up of carbon or platinum.A DC
voltage of about 8-15V/cm length of paper is normally applied.
•Types of Paper Electrophoresis :There are two types of Paper
electrophoresis based on the voltage applied:
•Low voltage electrophoresis(about 100-300V)
•High voltage electrophoresis(1000V)
COMPONENTS
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11. •Buffer:
•Buffers of different pH and ionic strength are used in separation process, pH
of buffer to be used depends upon the types of compounds to be separated.
•The ionic strength (IS) of the buffer used in paper electrophoresis affects the
migration velocity compounds.
•Migration of compounds is inversely proportional to the ionic strength.
•Allow ionic strength, migration is faster, but the separated bands appear
diffused. Usually in strengths (IS) of 0.05-05 is used in most separations.
•Examples:
•Barbitone buffer (Verona’ buffer) (0.07 mole/litre, pH 8.6), Citrate buffer (0.07
mole/litre, pH 3.0 or pH 6.8).
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12. TYPES OF PAPER ELECTROPHORESIS
•Based on the intrument degin:
•Horizontal paper electrophoresis
•Vertical paper electrophoresis
•Continuous paper electrophoresis
•Horizontal and Vertical types are used in analytical scale, where as continuous
electrophoresis is tased on a preparative scale ie. A large quantity of sample
mixture is separated into individual compounds and they are used for different
purposes.
•The principles of all types are same, but the design of each instrument varies.
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13. HORIZONTAL PAPER ELECTROPHORESIS
•In horizontal paper electrophoresis Buffer solution of known pH and ionic
strength is filled in two beakers. Whatman filter paper of suitable grade and
convenient dimensions are cut and immersed in buffer solution.Sample
solution is applied at the centre of the paper and fixed in position.
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14. VERTICAL PAPER ELECTROPHORESIS
•Themigration of ions takes place by gravity. After sufficient migration, the
paper is taken out and dried, to fix the spots .
•Then the spots can be visualized like in paper chromatography. Quantitation of
spots can also be carried out using densitometer.

15. CONTINUOUS ELECTROPHORESIS
•In this type predetermined sample volume through a valve device is applied
continuously on the centre of paper.
•Suitable Voltage causes migrations of samples and compound separated as
bands. Thus each band is made to fall down and pure compounds are
collected in separate containers.
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16. 16

17. APPLICATION
•Clinical applications of paper electrophoresis include
study of sickle cell disease, hemoglobin abnormalities, and
separation of blood clotting factors and serum plasma
proteins from blood sample.
•It has also been used in separation and identification of
alkaloids, also be used for testing water samples, toxicity
of water, and other environmental components.
•Drug-testing industry uses paper electrophoresis to
determine presence of illegal drugs crime suspects.
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18. 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,etc.
•This is the most commonly used electrophoresis in biotechnology laboratories.
•Types of gels:
•Agarose gel: For separating larger nucleic acids.
•Polyacrylamide gel: For separating smaller nucleic acids. -SDS-PAGE(sodium
dodecyl sulphate-polyacrylamide GE):for separating the proteins.
•Starch: Non-separated proteins can be separated according to charge and size.
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19. PRINCIPAL
•When a potential difference is applied across the electrodes of a
horizontal electrophoretic tankcontaining agarose gel and
biomolecules (such as nucleic acids), then they get separated
according to their molecular sizeand 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.
•There are 2 types : HGE and VGE.
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20. 20

21. HORIZONTAL
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22. VERTICAL GEL ELECTROPHORESIS
•Acrylamide gel only used in vertical gel electrophoresis.
•Acrylamide gels have smaller pores (10 to 200 nm in diameter).
•It is more complex then HGE.
•SDS-PAGE (sodium dodecyl sulphate–polyacrylamide gel electrophoresis)
is a discontinuous electrophoretic system
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24. APPLICATION
•In Forensics: Gel electrophoresis can be used in the field of forensics to
identify samples of DNA.
•For Nanoparticles: A novel application for gel electrophoresis is to separate
or characterize metal or metal oxide nanoparticles (e.g. Au, Ag, ZnO, SiO2).
•Proteins are usually analyzed by SDS-PAGE.
•To check DNA Cloning
•For PCR Testing
•For Paternity Testing
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25. REFERENCE
•www.aesociety.org• http://www.namrata.co•
http://www.biotechnologynotes.com
•Principles of Instrumental Analysis –Doglas A Skoog, F. James Holler,
Timothy A. Nieman, 5
th
edition, Eastern press, Bangalore, 1998.
•Instrumental methods of analysis –Willards, 7
th
edition, CBS
publishers.
•https://en.m.wikipedia.org/wiki/Gel_electrophoresis
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26. THANK YOU
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