Electrophoresis Techniques

MOHAMMED AL-ZUBAIDI, PhD

 Theory of electrophoresis
• Introduction:
Electrophoresis is a physical method of analysis
which involves separation of the compounds that
are capable of acquiring electric charge in
conducting electrodes.
• Definition:
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
tends to travel towards the electrodes that bears
opposite charges.
 Theory of electrophoresis
The rate of migration of an ion in electrical field
depend on factors:
1. Net charge of molecule
2. Size and shape of particle
3. Strength of electrical field
4. Properties of supporting medium
5. Temperature of operation
 Theory of electrophoresis
Mobility
• Under the electrical field, the mobility of the
particle is determined by two factors:
1- Its charge
2- Frictional coefficient
• Size and shape of the particle decide the
velocity with which the particle will migrate
under the given electrical field and the
medium.
 Theory of electrophoresis
Strength of electrical field
• It determined by the force exerted on the particle, and the
charge the particle carrying.

F=QV
when force is exerted
on the particle it start
moving, however the
movement is restricted
by the experience of
the frictional force
because of the viscosity.
 Theory of electrophoresis
Effect of pH on Mobility
• As the molecule exist as amphoteric , they will
carry the charges based on the solvent pH.
• Their overall net charge is NEUTRAL when it is at
zwitter ion state. And hence the mobility is
retarded to zero.
• Mobility is directly proportional to the magnitude
of the charge, which is functional of the pH of
solvent.
• The pH is maintained by the use of Buffers of
different pH.
 Theory of electrophoresis
Electrophoretic velocity depends on:
Inherent Factors External environment
 Magnitude of its charge  Solution pH
 Charge density  Electric field
 Molecular weight  Solution viscosity
 Tertiary or quaternary  temperature
structure (i.e., its shape).
 Electrophoresis
• A separation technique
• Simple, rapid and highly sensitive
• used in clinical laboratories to separate charged
molecules from each other in presence of electric
field
 Serum Protein Electrophoresis
 Lipoprotein Analysis
 Diagnosis of Haemoglobinopathies and
Haemoglobin A1c
 Clinical applications of
Electrophoresis
 Determination of Serum Protein Phenotypes and
Micro heterogeneities e.g. α1- antitrypsin
deficiency, MM
 Genotyping of Proteins eg. ApoE analysis for
Alzheimer’s disease (polymorphic protein)
 Small Molecules (Drugs, Steroids) Monitoring
 Cerebrospinal Fluid Analysis
 Urine Analysis ( determination of
glomerulonephritis)
 Nucleic acids: DNA, RNA
 Types of electrophoresis
Zone electrophoresis
a) Paper electrophoresis
b) Gel electrophoresis
c) Thin layer electrophoresis
d) Cellulose acetate electrophoresis

Moving Boundary Electrophoresis

a) Capillary Electrophoresis
b) Isotachophoresis
c) Isoelectric Focusing
d) Immuno Electrophoresis
 Zone electrophoresis
• It involves the migration of the charged
particle on the supporting media (Paper,
cellulose acetate membrane, starch gel, poly
acrylamide).
• The separated components are distributed
into discrete zone on the support media.
• Supporting media is saturated with buffer
solution, small volume of the sample is
applied as narrow band.
 Zone electrophoresis
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.
 General method of operation
• Saturation of the medium with the buffer.
• Sample application.
• Electrophoretic separation.
• Removal of the supporting media.
 Instrumentation
• Electrophoresis chamber.
• Electrodes.
• Diffusion barriers.
• Supporting / stabilizing media. (inert to
sample and to any developing reagents)
 Paper electrophoresis

1. Horizontal paper Electrophoresis

2. Vertical paper Electrophoresis
 Paper electrophoresis
• Filter paper such as Whatmann no1 and no3 in strip of 3
mm or 5 cm wide have been used to good effect.
• Separation takes place in 12 to 14 hrs.

Advantages
• It is economical
• Easy to use

Disadvantages
• Certain compounds such as proteins, hydrophilic molecules
cannot be resolved due to the adsorptive and ionogenic
properties of paper which results in tailing and distortion of
component bands.
• Electro osmosis
 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”
• Gel is a colloid in a solid form (99% is water).
• It is important that the support media is electrically neutral.
• 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 basic disaccharide repeating units of 3,6-
anhydro-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
 Agar and agarose gel
Advantages
• Easy to prepare and small concentration of agar is required.
• Resolution is superior to that of filter paper.
• Large quantities of proteins can be separated and recovered.
• Adsorption of negatively charged protein molecule is negligible.
• It adsorbs proteins relatively less when compared to other medium.
• Sharp zones are obtained due to less adsorption.
• Recovery of protein is good, good method for preparative purpose

Disadvantages
• Electro osmosis is high.
• Resolution is less compared to polyacrylamide gels.
• Different sources and batches of agar tend to give different results and
purification is often necessary.

Application
• Widely used in immuno electrophoresis
 Polyacrylamide gel electrophoresis
(PAGE)
• It is prepared by polymerizing acryl amide 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 are stable over wide range of pH
and temperature.
• Gels of different pore size can be formed.
• Simple and separation speed is good comparatively.
 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
 PAGE-procedure
• The gel of different pore sizes is cast into a column inside a vertical
tube, often with large pore gel at the top and small pore gel at the
bottom.
• Microgram quantity of the sample is placed over the top of the gel
column and covered by a buffer solution having such a pH so as to
change sample components into anions.
• The foot of the gel column is made to dip in the same buffer in the
bottom reservoir.
• Cathode and anode are kept above and below the column to
impose an electric field through the column.
• Macromolecular anions move towards the anode down the gel
column.
• There is no external solvent space, all the migratory particles have
to pass through the gel pores.
• Rate of migration depends on the charge to mass ratio.
• Different sample components get separated into discrete migratory
bands along the gel column on the basis of electrophoretic mobility
and gel filtration effect.
 SDS-polyacrylamide gel
electrophoresis (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.
• When a detergent SDS added to PAGE the combined procedure is termed as
SDS PAGE.
• 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.
• 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 attracted toward an anode in an electric field.
 Sodium dodecyl sulfate (SDS-PAGE)
• Native protein is unfolded by heating in the presence
of mercaptoethanol and SDS.
• SDS binds to the protein so that it stays in solution and
denatures.
• Large polypeptides bind more SDS than small
polypeptides, so proteins end up with negative charge
in relation to their size.
• When treated with SDS and a reducing agent, the
polypeptides become rods of negative charges with
equal “charge densities" or charge per unit length.
• Thus, we can separate the proteins based on their mass
 Starch
• A suspension of granular starch should be boiled in a buffer to give
a clear colloidal suspension.
• The suspension on cooling sets as a semisolid gel due to
intertwining of the branched chains of amylopectin.
• In order to avoid swelling and shrinking petroleum jelly is used.

Advantages
• High resolving power and sharp zones are obtained.
• The components resolved can be recovered in reasonable yield
especially proteins.
• Can be used for analytical as well as preparative electrophoresis.

Disadvantages
• Electro osmotic effect.
• Variation in pore size from batch to batch
 Thin layer electrophoresis
Studies can be carried out in thin layer of silica,
alumina.

Advantages
• Less time consuming and good resolution.

Application
• Widely used in combined electrophoretic-
chromatography studies in two dimensional study
of proteins and nucleic acid hydrolysates
 Cellular acetate electrophoresis
• 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 for staining glycoproteins.

Advantages
• No tailing of proteins or hydrophilic materials.
• Available in wide range of particle size and layer thickness.
• Give sharp bands and offer good resolution.
• High voltage can be applied which will enhance the resolution.

Disadvantages
• Expensive.
• Presence of sulphonic and carboxylic residue causes induced electro osmosis during
electrophoresis.

Application
• Widely used in analysis of clinical and biological protein samples (albumin and
globulins).
• Alternative to paper electrophoresis.
Moving boundary electrophoresis
PRINCIPLE:
The moving boundary method allows the
charged species to migrate in a free moving
solution without the supporting medium
Moving boundary electrophoresis
Advantages
• 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.

Application
• To study homogenecity of a macromolecular system.
• Analysis of complex biological mixtures
 Capillary electrophoresis
• The principle behind electrophoresis is that charged molecules will migrate toward
the opposite pole and separate from each other based on physical characteristics.
• Capillary electrophoresis has grown to become a collection of a range of
separation techniques which involve the application of high voltages across buffer
filled capillaries to achieve separations .
• Capillary electrophoresis, then, is the technique of performing electrophoresis in
buffer filled, narrow-bore capillaries, normally from 25 to 100 mm in internal
diameter (ID).
• A high voltage (typically 10-30 kV) is applied.
• Capillaries are typically of 50 μm inner diameter and 0.5 to 1 m in length.
• Due to electro osmotic flow, all sample components migrate towards the negative
electrode.
• The capillary can also be filled with a gel, which eliminates the electro osmotic
flow. Separation is accomplished as in conventional gel electrophoresis but the
capillary allows higher resolution, greater sensitivity, and on-line detection.
• The capillary is filled with electrolyte solution which conducts current through the
inside of the capillary. The ends of the capillary are dipped into reservoirs filled
with the electrolyte.
• Electrodes (platinum) are inserted into the electrolyte reservoirs to complete the
electrical circuit.
Electroosmotic flow
The surface of the silicate glass capillary contains negatively-
charged functional groups that attract positively-charged
counter ions. The positively-charged ions migrate towards the
negative electrode and carry solvent molecules in the same
direction. This overall solvent movement is called electro
osmotic flow. During a separation, uncharged molecules move
at the same velocity as the electro osmotic flow (with very
little separation). Positively-charged ions move faster and
negatively-charged ions move slower.

A small volume of sample is moved into one end of the

capillary. The capillary passes through a detector, (usually a
UV absorbance detector), at the opposite end of the capillary.
• Application of a voltage causes movement of sample ions
towards their appropriate electrode usually passing
through the detector.
• A plot of detector response with time is generated which is
termed an electropherogram
 Isotachophoresis
The technique of isotachophoresis depends on the development of
potential gradient.

Principle:
• Based on principle of moving boundary electrophoresis. A leading
electrolyte(e.g. chloride) with a higher mobility than the analytes, and a
trailing electrolyte(e.g. glycinate) with a lower mobility are used.
• Solution in which the separation takes place is normally an aqueous
medium, which contains sucrose to provide a higher density to the
solution.
• Where the separation by Isoelectric focusing depends on the existence of
a pH gradient in the system. The technique of Isotachophoresis depends
on the development of a potential gradient.
• Separation of the ionic components of the sample is achieved through
stacking them into discrete zones in order of their mobilities , producing
very high resolution.
 Isotachophoresis
• The analyte are positioned between the electrolytes and, when the voltage is
applied, they migrate in order of decreasing mobility.
• This establishes the potential gradient; from that point on, all the analyte move
at the same speed.
• Individual zones border one another but represent completely separated
components without overlap.
• In isotachophoresis no background electrolyte(buffer) is mixed with the sample,
so current flow is carried only by charged sample ions.
• Once a faster moving component separates completely from a slower moving
one, It creates a region of depleted charge between the two that increases the
resistance and therefore local voltage in that region.
• This increased voltage causes the slower component to migrate faster and close
the gap, thereby concentrating it and increasing the conductivity of its zone until
it matches that of the faster ion.
• Ultimately all ions migration at the rate of the faster ion in the zones that differ in
thickness, depending on their original concentrations.

Application
• Isotachophoresis that been used for the separation of proteins as well as
inorganic substances.
 Isoelectric focusing
• All proteins have an isoelectric point pH .
• 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 procedure to determine the isoelectric point (PI) of proteins thus, a mixture of proteins can
be electrophorised 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 (600-900D) 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.
Advantages
• As spreading of bands is minimized due to application of the
applied field and the PH gradient , high resolution can be achieved.
• Proteins that differ by as little as 0.001 PH units can be separated.

Disadvantages
• Because carrier ampholytes are generally used in high
concentration, a high voltage (up to 2000v ) 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 , immunology, cytology and taxonomy.