Electrophoresis

Dr. Thamilarasi K
Senior Scientist
Agri-bioresources Augmentation Division
ICAR – National Institute of Secondary Agriculture
Namkum, Ranchi
Introduction

• Electrophoresis may be defined as the migration of the charged particle through a solution
under the influence of an external electrical field. Ions that are suspended between two
electrodes tend to travel towards the electrode that bears opposite charges.
• Electrophoresis is a laboratory technique used to separate DNA, RNA, or protein molecules
based on their size and electrical charge.
• An electric current is used to move molecules to be separated through a gel or suitable matrix.
• Pores in the gel work like a sieve, allowing smaller molecules to move faster than larger
molecules.
• The conditions used during electrophoresis can be adjusted to separate molecules in a desired
size range.
Theory of electrophoresis
• Movement of ions or their mobility depends upon the frictional coefficient, which in turn depends on
the function of some of the physical properties of the molecules such as weight, molecular shape, size
etc.
• The law of electrostatics: F electric = qE
where, F electric is electrical force on an ion,
q is the charge on the ion,
E is the electric field strength.
The resulting electrophoretic migration of the ion though the solution is opposed by a frictional force
F friction = Vf
where, V is velocity (rate of migration) of the ion and
f is its ‘frictional coefficient’.
The frictional coefficient is a measure of the drag that the solution exerts on the moving ion and is
dependent on the size, shape and state of the ion as well as on the viscosity of the solution.
Principle

• The working principle of electrophoresis is that it causes the

separation of the molecules, ions or colloidal particles that suspends
in the matrix under the force of an electric field.
• When charged molecules are placed in an electric field, they migrate
toward either the positive or negative pole according to their charge.
• The electric field allows the migration of the positively charged
molecule towards the cathode and the migration of negatively
charged molecule towards the anode.
• Therefore, electrophoresis is the electrokinetic phenomenon where
the motion of molecules occurs under an electric field.
Rate of migration

The rate of migration (Separation of particles) during electrophoresis

will depend on the following factors:

1. Net charge of the molecule

2. Size and shape of the molecule
3. Strength of the electric field
4. Properties of the supporting medium
5. Temperature and pH of operation
Applications of electrophoresis

• It is a tool of macromolecular separation.

• Many biological complex samples can be separated by using various
methods of electrophoresis.
• In some cases, identification of molecules is also possible.
• Gel method is more commonly used for routine laboratory
experiments as well as research oriented separations and
identification.
• Electrophoresis is handy tool for biologist and biochemist like the use
of chromatographic techniques by organic chemist.
Types of electrophoresis

Zone electrophoresis : involves the migration of charged molecules with the presence of a
supporting medium
• Paper electrophoresis
• Gel electrophoresis
• Thin layer electrophoresis
• Cellulose acetate electrophoresis
Moving boundary electrophoresis: involves the migration of charged molecules in a free
moving solution, without the presence of a supporting medium
• Capillary electrophoresis
• Isotachophoresis
• Isoelectric Focusing
• Immuno-electrophoresis
Zone electrophoresis
• It involves the migration of the charged particle on the supporting media (Paper, cellulose acetate
membrane, starch gel, poly acrylamide).
• Supporting media is saturated with buffer solution, small volume of the sample is applied to the
supporting medium.
• The separated components are distributed into discrete zone on the support media.

Advantages
• Useful in biochemical investigations.
• Small quantity of sample can be analyzed.
• Low cost and easy maintenance.

Disadvantages
• Unsuitable for accurate mobility and isoelectric point determination.
• Due to the presence of supporting medium, technical complications such as capillary flow,
electro osmosis, adsorption and molecular sieving are introduced.
Paper electrophoresis
Paper electrophoresis (PE) is useful for the separation of small-charged molecules, such as amino acids
and small proteins using a strip of paper (chromatography paper).
Applications of Paper Electrophoresis
• A simple, inexpensive, and accurate laboratory procedure for various research and clinical studies.
• Easily available and easy to handle, allowing new methodologies to be tried and developed with
convenience.
• Clinical applications of PE include study of sickle cell disease, hemoglobin abnormalities, and
separation of blood clotting factors and serum plasma proteins from blood sample.
• Used in separation and identification of alkaloids.
• Drug-testing industry uses paper electrophoresis to determine presence of illegal or recreational
drugs in job applicants and crime suspects.
• Since 1950s used by the investigators and in forensics to analyze inks used in currency to check the
counterfeiters.

Lack of sensitivity and reproducibility

are limitations of PE
 Cellulose Acetate Electrophoresis
• Many biological samples adsorb on cellulose (that is paper); the adsorption is because of
hydroxyl groups present in cellulose. Adsorption reduces the movement and therefore
causes tailing of spots/bands. This spreading of spots reduces resolution.
• To solve this problem cellulose acetate membrane is used where most of the hydroxyls
have been converted acetate groups.
• Cellulose acetate is preferred because of its simplicity and high resolution at low applied
voltage.
• It contains 2-3 acetyl groups per glucose unit and its adsorption capacity is less than that
of paper.
• It gives sharper bands.
• Provides a good background or staining glycoprotein
Applications
• Widely used in analysis of clinical and biological protein sample (albumin and globulins).
• Alternative to paper electrophoresis.
Gel electrophoresis

• Separation is brought about through molecular sieving technique, based on the

molecular size of the substances.
• Gel material acts as a “molecular sieve”
• It is important that the support media is electrically neutral and gel is a colloid and
essentially 99% water.
• Different types of gels which can be used are; Agar and Agarose gel, Starch,
Sephadex, Polyacrylamide gels.
• A porous gel acts as a sieve by retarding or, in some cases, by completely
obstructing the movement of macromolecules while allowing smaller molecules to
migrate freely.
Agar and agarose gel

• Agar is a mixture of polysaccharides extracted from sea weeds. Agarose is a highly purified
uncharged polysaccharide derived from agar.
• Agarose is chemically disaccharide i.e., a copolymer of 1,3-linked β-D-galactose and 1,4-
linked 3,6-anhydro-α-L-galactose.
• Agarose dissolves when added to boiling liquid. It remains in a liquid state until the
temperature is lowered to about 40 °C at which point it gels.
• The pore size may be predetermined by adjusting the concentration of agarose in the gel.
Agarose gels are fragile. They are actually hydrocolloids, and they are held together by the
formation of weak hydrogen and hydrophobic bonds.
• The pores of an agarose gel are large, agarose is used to separate macromolecules such as
nucleic acids, large proteins and protein complexes.
Agarose gel
electrophoresis
 Advantages of Agarose gel electrophoresis
• Accuracy: Agarose gel electrophoresis is a dependable and precise method for sorting DNA fragments based on
their size. It enables scientists to determine the presence or absence of specific DNA fragments and estimate their
size range.
• Reliability: Agarose gel electrophoresis is a commonly used and well-established technique in molecular biology. It
has been thoroughly tested and is regarded as a reliable approach for DNA analysis.
• Reproducibility: When standardised techniques and quality control measures are followed, agarose gel
electrophoresis is a highly reproducible technology. This ensures that results are consistent and comparable
between studies and laboratories.
• Cost-Effectiveness: When compared to other gel matrices used in electrophoresis, such as polyacrylamide gels,
agarose is a cost-effective material. Agarose gel electrophoresis is a low-cost method for analysing DNA since it is
commonly available.
• Simple and Quick Preparation: Agarose gels are relatively easy and quick to make. They simply require a single-
component agarose and do not require polymerization catalysts. The gel is simple to pour and solidifies at room
temperature without denaturing the DNA samples.
• Sample Recovery: After separation, agarose gel electrophoresis enables for the recovery of DNA samples. The
desired DNA bands can be excised from the gel for further downstream applications such as DNA purification,
 Disadvantages of Agarose Gel Electrophoresis

• Concerns about safety: Agarose gel electrophoresis uses potentially hazardous chemical
(ethidium bromide), and heating procedures, which can be dangerous if not handled
appropriately. Adequate safety procedures and measures should be followed to safeguard
the safety of researchers.
• Inaccurate size determination: Agarose gel electrophoresis only provides a rough
estimate of DNA fragment sizes. The gel’s resolution is restricted, making it difficult to
properly measure the size of DNA pieces. Accurate size measurement necessitates the
use of additional procedures, such as DNA sequencing or other specialised techniques.
• Resolution is less compared to polyacrylamide gels.
• Different forms of genetic material may run in unpredictable forms.
 Applications of Agarose gel
electrophoresis

• Agarose gel electrophoresis is a routinely used method for separating DNA or RNA.
• A very handy technique to find the approximate molecular weight of DNA.
• 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.
• Agarose gel electrophoresis is commonly used to resolve circular DNA with different
supercoiling topology, and to resolve fragments that differ due to DNA synthesis.
• 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.
Polyacrylamide gel electrophoresis (PAGE)
• It is prepared by polymerizing acrylamide monomers in the presence of methylene-bis-acrylamide
to cross link the monomers. Structure of acrylamide (CH2=CH-CO-NH2).
• Polyacrylamide gel structure held together by covalent cross-links.
• Polyacrylamide gels are tougher than agarose gels.
• It is thermostable, transparent, strong and relatively chemically inert.
• Gels are uncharged and are prepared in a variety of pore sizes.
• Proteins are separated on the basis of charge to mass ratio and molecular size, a phenomenon
called molecular sieving.
Advantage
• Gels of different pore size can be formed which are stable over wide range of pH and
temperature.
• Resolution is much better than agarose gels and used to separate smaller DNA molecules which
are difficult to be separated in agarose gels.
Types of PAGE
NATIVE-PAGE
• Native gels are run in non-denaturing conditions, so that the analyte's natural
structure is maintained.
• Separation is based upon charge, size, and shape of macromolecules. Useful for
separation or purification of mixture of proteins.
• This was the original mode of electrophoresis.
DENATURED-PAGE OR SDS-PAGE
• Separation is based upon the molecular weight of proteins.
• The common method for determining MW of proteins.
• Very useful for checking purity of protein samples.
SDS-PAGE
• SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique widely used
in biochemistry, forensics, genetics and molecular biology to separate proteins according to their
electrophoretic mobility.
• SDS coats protein molecules giving all proteins a constant charge-mass ratio.
• Due to masking of charges of proteins by the large negative charge on SDS binding with them, the
proteins migrate along the gel in order of increasing sizes or molecular weights.
• SDS is an anionic detergent which denatures secondary and non–disulfide– linked tertiary
structures by wrapping around the polypeptide backbone. In so doing, SDS confers a net negative
charge to the polypeptide in proportion to its length. Mercaptoethanol is used to break
disulphide links.
• Molecules in solution with SDS have a net negative charge within a wide pH range.
• A polypeptide chain binds amounts of SDS in proportion to its relative molecular mass.
• The negative charges on SDS destroy most of the complex structure of proteins, and are strongly
Moving Boundary
Advantages
Electrophoresis • Biologically active fractions can be
recovered without the use of denaturing
agents.
• A reference method for measuring
electrophoretic motilities.
Disadvantages
• Costly
• Elaborate optical system are required.
Applications
• To study homogeneity of a
macromolecular system.
• Analysis of complex biological mixtures
Moving Boundary Electrophoresis
• Capillary electrophoresis: is a type of liquid phase micro-separation analysis technology
that uses capillary as the separation channel, high-voltage direct current electric field as
the driving force, and various characteristics of the sample as the basis. CE can be used to
analyze components ranging from organic ions to biological macromolecules such as
proteins and nucleic acids.
• Isotachophoresis (ITP) is a technique in analytical chemistry used for selective separation
and concentration of ionic analytes. It is a form of electrophoresis; charged analytes are
separated based on ionic mobility, a quantity which tells how fast an ion migrates through
an electric field. Separation is obtained by using discontinuous buffer system.
• Immunoelectrophoresis is a general name for a number of biochemical methods for
separation and characterization of proteins based on electrophoresis and reaction with
antibodies.
Isoelectric focusing
 All proteins have an isoelectric point pH . The isoelectric point (pI) is the pH of a solution at which the net charge of a
protein becomes zero.

 When electrophoresis is run in a solution buffered at constant pH, proteins having a net charge will migrate towards the
opposite electrode so long as the current flows.

 The use of pH gradient across the supporting medium causes each protein to migrate to an area of specific pH. The pH of
the protein equals the pH of the gradient, thus resulting in sharp well defined protein bands.

 A mixture of proteins can be electrophorized through a solution having a stable pH gradient from the anode to the cathode
and each protein will migrate to the position in the pH gradient according to its isoelectric point. This is called isoelectric
focusing.

 Protein migrate into the point where its net charge is zero – isoelectric pH.

 Protein is positively charged in solutions at pH below its PI and will migrate towards the cathode.

 Protein is negatively charged in solution at pH above its PI will migrate towards the anode.

 They will be in the Zwitter ion form with no net charge so the further movement will cease.

 Ampholytes (amphoteric electrolytes)- low molecular mass (600-900 D) oligomers with aliphatic amino and carboxylic acid
groups with a range of isoelectric points. Ampholytes help maintain the pH gradient in the presence of high voltage. Can
also use gels with immobilized pH gradients - made of acrylamide derivatives that are covalently linked to ampholytes.
Isoelectric focusing
Advantages
• As spreading of bands is minimized due to application of the applied field and the
pH gradient , high resolution can be achieved.
Disadvantages
• Since carrier ampholytes are generally used in high concentration, a high voltage
(up to 2000 V ) is necessary. As a result, the electrophoretic matrix must be cooled
which sometimes makes it difficult.
Applications
• For separating proteins and peptides.
• For research in enzymology and immunology.
2-D gel electrophoresis

• The complex biological samples are efficiently resolved in 2-D gel electrophoresis.
• It involves the combination of charge and molecular weight to provide much greater
separation in comparison to use of the individual property.
• The 2-D electrophoresis is a combination of isoelectic focusing (IEF) followed by
SDS-PAGE in a perpendicular direction.
• The isoelectric focusing separates the protein sample based on their isoelectric pH
(it is indirectly related to the charge present on the protein) where as SDS-PAGE
separates the molecules based on size (it is indirectly related to the molecular
weight).
• In general analysis of complex bacterial lysate or tissue extract produces 1000-2500
well separated spots. With a sensitive detection tool and image analysis software,
individual can be identified and compared under different conditions.