2. Introduction
• Electrophoresis may be defined as migration of charged particles through
solution under the influence of an external electric field.
• Ions having positive charge migrate towards a negative electrode and ions
with negative charge migrate toward a positive electrode.
• Ions have different migration rates depending on their total charge, size &
shape. So they can be separated.
• More highly charged ions and ions with smaller size ( higher charge to size
ratio) migrate at faster rate than ions with large size and low charge.
3. • The principle of electrophoresis is when electricity is applied to the medium containing biological
molecules, depending on their net charge & molecular size, they migrate differentially. Thus diff. Proteins/
DNA can be separated .
The force created by electric field can be determined by F = EQ. F is the force, E is electric field & Q is the
charge on molecule or ion.
Thus, more is the charge more is the force on molecules with same size.
4. Types of Electrophoresis
1. Zone Electrophoresis
• Paper electrophoresis
• Gel electrophoresis
• Thin layer electrophoresis
• Cellulose acetate electrophoresis
2. Free Electrophoresis
• Moving boundary electrophoresis
• Capillary electrophoresis • Isoelectric focusing
• Isotachophoresis • Immuno electrophoresis
5. Zone Electrophoresis
• It involves migration of the charged particle on the supporting media.
• Paper, gel, cellulose acetate membrane, starch, poly acrylamide.
• The components that are separated are distributed into discrete zones on
supporting media.
• Supporting media is saturated with buffer solution, small amount of sample
is applied as narrow band .
• Substances separated are nucleic acid, protein, bio polymers .
6. Advantages & Disadvantages
• Easy maintenance & low cost
• Useful in biochemical investigations
• Small amount of sample can be Analyzed
• Unsuitable for accurate mobility and iso electric point determination
• General Method Of Operation :
• Initially Saturation of media with buffer followed by sample application and
electrophoretic separation & then removal of supporting media.
8. 1. Paper Electrophoresis
• In this, sample to be separated is applied to a strip of paper moisturised using a
kind of buffer solution. Eg. Diethyl barbituric acid & barbituric acid dissolved
in alkali, pH 8.6 .
• There are separate tanks of this buffer solution and each end of paper is dipped
in these tanks.
• Different types of cathode and anode are used.
• An electric current is applied, which forces the sample to move on direction of
electrode with opposite polarity.
• Separation takes place in 12 to 14 hours.
9. Principle
• When charged molecules are placed in an electric field, they migrate towards
either the positive or negative electrode according to their charges. In contrast
to proteins, which can have either a net positive or net negative negative
charge, nucleic acids have a consistent negative charge imparted by their
phosphate backbone, & migrate towards anode .
• Equipments : Power pack - It provides a stable direct current & has control for
both voltage & current output.
Electrophoretic cell : It contains electrodes, buffer reservoirs, a support for
paper & a transparent insulating cover. Electrodes are usually made of platinum.
10. Procedure
1. A long strip of filter paper is moistened with a suitable buffer solution of desired pH
& the sample is applied transversely across the central part of strip.
2. Ends are fixed to dip in buffer solution in 2 troughs fitted with electrodes.
3. Electric field of about 20 volts/cm is established.
4. The charged particles of sample migrate along the strip towards respective electrodes
of opposite polarity, according to net charges, sizes & interaction with solid matrix.
5. Homogenous group of particles migrate as separate band.
6. Electrophoresis is carried out for 16 - 18 hours.
7. Proteins are stained ( bromophenol blue ) to make them visible.
8. The separated bands appear as distinct band.
11. 1. Advantages : It is economical.
• It is easy to use.
2. Disadvantages : Certain compounds such as protein, hydrophilic
molecules can’t be resolved due to adsorptive and ionogenic properties of
paper which results in tailing and distortion of components bands.
• Electro osmosis.
Types of Paper Electrophoresis: Horizontal paper Electrophoresis
Vertical Paper Electrophoresis
12.
13. In horizontal paper electrophoresis, the
gel is cast horizontally & placed in
chamber ( filled with running buffer )
that is divided into two sections with
gel in the middle, forming positive
charge at one end & negative at
another.
A continuous running buffer is used
in horizontal gel electrophoresis.
14. Vertical gel electrophoresis contains stacking gel
and resolving gel. The stacking gel concentrates
proteins that are loaded into the well so that the
proteins can start to migrate at the same time. After
stacking, the resolution gel separate proteins based
on the molecular size.
It can give important diagnostic information
concerning serum proteins, and is invaluable
in the differential diagnosis of diseases in
which there are abnormal hemoglobins.
15. Application
1. It is simple, inexpensive & accurate laboratory procedure for various research & clinical
studies.
2. Clinically it is used to study sick cell anaemia, haemoglobin abnormalities & separation of
blood clotting factors & serum plasma protein from blood sample.
3. It is also used in separation & identification of alkaloids.
4. Paper Electrophoresis can also be used for testing water samples, toxicity of water & other
environmental components.
5. Drug - testing industry uses this technique to determine presence of illegal drugs crime
suspects.
6. Separation of amino acids, protein serum & anti biotic .
16. 2. Gel Electrophoresis
• In this, separation is brought about through molecular sieving technique based on
molecular size of substance.
• Gel material acts as as molecular sieve.
• Gel is a colloid in a solid form ( 99% in water ).
• Support media should be electrically neutral.
• Different types of gels which can be used are agar, agarose gel, starch & polyacrylamide
gels ( most common due to high stability).
• The gel acts as a sieve by reducing by movement of macromolecules and allowing micro
molecules to move freely and are used to separate DNA, RNA & Proteins.
17. Agar and Agarose Gel
• Agar is a mixture of polysaccharides extracted from sea weed.
• Agarose is a highly purified uncharged polysaccharide derivative of agar.
• Agarose is chemically basic disaccharide repeating units of 3,6 – Anhydro-L- Galactose.
• Agarose dissolves in boiling liquid. It remains liquid until the temperature lowered to about 40
degree Celsius.
• Pore size may be pre determined by adding the concentration agarose in gel.
• Agarose gels are fragile as they are held together by weak H bonds.
• Pores of agarose gel are large. It is used to seperate macromolecules such as nucleic acid, large
proteins ( albumin ) & protein complex.
18. Polyacrylamide Gel Electrophoresis (PAGE)
• It is prepared by polymerising acrylamide monomer in presence of Methylene- Bis-
Acrylamide to crosslink the monomer ( covalent crosslink)
• Polyacrylamide gel are tougher than agarose gel.
• It is thermostable, transparent, strong & relatively chemically inert.
• PAGE is used in forensic chemistry, biochemistry, genetics, molecular biology &
biotechnology to separate biological macromolecules, usually protein & nucleic acid.
• Protein are separated by molecular sieving ( based on charge to mass ratio and molecular
size)
19. • Advantages: Polyacrylamide gels are stable over wide range of PH &
temperature.
• Simple & separation speed is good enough.
• Types of PAGE : 1. Native PAGE
2. Denatured PAGE or SDS- PAGE
1. Native PAGE : Native gels are run in non denaturing conditions, so that the
analyte natural structure is maintained. Separation is based upon charge, size and
shape of macromolecules.
Useful for separation and purification of mixture of proteins.
2. SDS PAGE : In this separation is based on molecular weight of Proteins.
Useful for checking purity of protein samples.
20. Principle
• It consists in the separation of molecules on the basis of their movement rate
through a gel under the influence of an electrical field. An electric current is
applied across the gel so that one end of the gel has a positive charge and the other
end has a negative charge. The movement of charged molecules is called migration.
Molecules migrate towards the opposite charge.
• Gel electrophoresis is a widely used technique for the analysis of DNA, nucleic acids
and proteins.
21. • Gel electrophoresis separates DNA fragments by size in a solid support medium such as
an agarose gel. Sample (DNA) are pipetted into the sample wells, followed by the
application of an electric current which causes the negatively-charged DNA to migrate
(electrophorese) towards the anodal, positive end. The rate of migration is proportional to
size: smaller fragments move more quickly and wind up at the bottom of the gel.
• DNA is visualised by including in the gel an intercalating dye, ethidium bromide. DNA
fragments take up the dye as they migrate through the gel. Illumination with ultraviolet
light causes the intercalated dye to fluoresce.
• The larger fragments fluoresce more intensely. Although each of the fragments of a
single class of molecule is present in equimolar proportions, the smaller fragments
include less mass of DNA, take up less dye, and therefore fluoresce less intensely. A
“ladder” set of DNA fragments of known size can be run simultaneously and used to
estimate the sizes of the other unknown fragments.
22. Equipments for Gel Electrophoresis
The equipment and supplies necessary for conducting agarose gel electrophoresis are
relatively simple and include:
1.An electrophoresis chamber and power supply
2.Gel casting trays, which are 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.
3.Sample combs, around which molten medium is poured to form sample wells in
the gel.
4.Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA
(TBE).
24. 5. Loading buffer, which contains something dense (e.g. glycerol) to allow
the sample to “fall” into the sample wells, and one or two tracking dyes,
which migrate in the gel and allow visual monitoring or how far the
electrophoresis has proceeded.
6. Staining: DNA molecules are easily visualised under an ultraviolet lamp
when electrphoresed in the presence of the extrinsic fluorescent ethidium
bromide. Alternatively, nucleic acids can be stained after electrophoretic
separation by soaking the gel in a solution of ethidium bromide. When
intercalated into double stranded DNA, fluorescence of this molecule
increases greatly. It is also possible to detect DNA with the extrinsic
fluorescent 8 -anilinonaphthalene -1- sulphonic acid.
7. Transilluminator (an ultraviolet light box), which is used to visualise
stained DNA in gels.
25.
26. Applications
Gel electrophoresis is a routinely used method for separating proteins, DNA or RNA.
• Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting
• Separation of restricted genomic DNA prior to Southern analysis, or of RNA prior to
Northern analysis.
• The agarose gel electrophoresis is widely employed to estimate the size of DNA
fragments after digesting with restriction enzymes, e.g. in restriction mapping of cloned
DNA.
• In the separation of DNA fragments for DNA fingerprinting to investigate crime scenes.
To analyse results of polymerase chain reaction. To analyse genes associated with a
particular illness. In DNA profiling for taxonomy studies to distinguish different species
27. Applications
• Agarose gel electrophoresis is commonly used to resolve circular DNA with
different supercoiling topology, and to resolve fragments that differ due to
DNA synthesis.
• In addition to providing an excellent medium for fragment size analyses,
agarose gels allow purification of DNA fragments. Since purification of
DNA fragments size separated in an agarose gel is necessary for a number
molecular techniques such as cloning, it is vital to be able to purify
fragments of interest from the gel.
