BCHEM 264

BIOPHYSICS
Lecture Four

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST
 THEORY AND METHODS OF
ELECTROPHORESIS
• What is electrophoresis?
• It is the motion of charged particles in a colloid under the influence of an electric
field
• Particles with positive charges move toward the cathode and those with a negative
charges move toward the anode
• The phenomenon was observed for the first time by Ferdinand Friedrich Reuss in
1807
• In his experiment, he found that application of constant electric field caused clay
particles dispersed in water to migrate at different speeds depending on their sizes
• Electrophoresis is therefore a technique for separating different types of molecules
based on their patterns (size, shape, charge) of movement in an electric field
• Many important biomolecules, such as amino acids, peptides, proteins, nucleic
acids possess ionizable groups. At any given pH, these exist in solution as
electrically charged particles. Under influence of electric field, the charged particles
migrate either to the cathode or to the anode, depending on their net charge

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 2
 WHAT IS ELECTROPHORESIS?

20XX PRESENTATION TITLE

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 3
THEORY AND
METHODS OF Power pack
ELECTROPHORESIS
Equipment required: Horizontal unit
1. A power pack
2. An electrophoresis unit

Electrophoresis units are

available for running either
vertical or horizontal gel
system
Electrophoresis unit
Vertical gel systems are routinely
used for protein separation on
acrylamide gels
Horizontal gel systems are used
for DNA separation on agarose
gels

4
Vertical unit
Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST
 EQUATIONS RELATING TO
ELECTROPHORESIS

• When a potential difference (voltage) is applied

across the electrodes, it generates an electric
gradient, E
• E is the applied voltage, V, divided by the distance,
d, between the electrodes (E=V/d)
• In the presence of this electric gradient E, the force
on a molecule bearing a charge of q coulombs is
given by Eq newtons.
• Eq is the force that drives a charged molecule
toward an electrode

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 5
 EQUATIONS RELATING TO
ELECTROPHORESIS
• As the charged particle moves toward the electrode, it encounters
frictional resistance imposed by the medium that retards its
movement
This frictional force is governed by the following four
characteristics of the medium and particle:
• Hydrodynamic size of particle
• Shape of the particle
• Pore size of the medium in which it is moving
• Viscosity of the buffer in which it is immersed
• The velocity, v, of a charged particle in an electric field is
therefore given by the force on the particle divided by the
resistance to movement, i.e.,
v =Eq/f
where f is the frictional coefficient
Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 6
 EQUATIONS RELATING TO
ELECTROPHORESIS

• A more common term used to describe the characteristic

movement of a particle in electrophoresis is
electrophoretic mobility, μ,
• μ is the ratio of the velocity of the charged particle to the
field strength (the electric gradient)
μ = v/E
• When a potential difference is applied, molecules with
different overall charges will begin to separate out owing
to their different electrophoretic mobilities
• Molecules with similar charges will begin to separate if
they have different sizes, since they will experience
different frictional forces
Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 7
 EQUATIONS RELATING TO
ELECTROPHORESIS

• Some forms of electrophoresis rely totally on the

different charges on the molecules to achieve separation
• Other methods exploit differences in molecular size and
therefore encourage frictional effect to bring about
separation
• Movement must be monitored to prevent particles from
moving beyond the electrodes
• By the time the electric field is removed, mixtures of
molecules would have moved at different velocities
toward the electrodes, thereby separation is achieved

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 8
EQUATIONS
RELATING TO
ELECTROPHORESIS

- • The separated samples are then

located by staining with an
appropriate dye or by
autoradiography if the sample is
radiolabeled

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 9
 SOME TECHNICAL PROBLEMS
WITH ELECTROPHORESIS-
HEATING
• The current in the solution between the electrodes is
conducted mainly by the buffer ions, with a small
proportion conducted by the sample ions
• Ohm’s law expresses the relationship between
current, I, voltage V, and resistance R
• Ohm’s law: V=IR
• Increase in applied voltage leads to increase in
current, hence acceleration of electrophoretic
separation

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 10
SOME TECHNICAL PROBLEMS WITH
ELECTROPHORESIS- HEATING
• The velocity of migration of particles, hence the
distance moved will be proportional to both current
and time
• Increase in current causes faster movement of
particles and ions, which leads to generation of heat,
a major problem in electrophoresis

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 11
 EQUATIONS RELATING TO
ELECTROPHORESIS

• During electrophoresis the power, W in watts, generated in the

support medium is given by
W I R 2

• Most of this power is dissipated as heat

• Heating of the electrophoretic medium produces the following
effects:
1. Increased rate of diffusion of sample and buffer ions leading to
broadening of separated samples
2. Formation of convection currents, which leads to mixing of separated
samples
3. Denaturation of thermally unstable samples, such as proteins,
4. Decrease in buffer viscosity, hence reduction in the resistance
of the medium
Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 12
 ELECTROPHORESIS AND HEATING
PROBLEMS

• Constant heat generation is a problem. One way to overcome

the heating problem is to run electrophoresis at very low
power, i.e. low current
• Running electrophoresis at low current slows down
movement of ions, long separation times are required, poor
resolution

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 13
 ELECTROPHORESIS AND HEATING
PROBLEMS
• Compromise conditions have to be sought, with reasonable power
settings in order to have acceptable separation times, and an
appropriate cooling system to remove liberated heat
• With the use of such systems, heating is not always totally
eliminated. With the use of cylindrical tubes or gel slabs, heat is
removed only from the edges, resulting in a temperature gradient
within the gel, with the center being hotter than the edges
• Since the warmer fluid at the center is less viscous, electrophoretic
mobilities are greater in this region. Note that for every 1°C rise in
temperature, electrophoretic mobility increases by 2%
• Electrophoretic zones develop a bowed shape, with the zone center
migrating faster than the edges

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 14
 ELECTROPHORESIS AND
ELECTROENDOSMOSIS
• Presence of charged groups on the surface of the
support medium establishes a phenomenon known
as electroendosmosis (or electroosmotic flow). For
example,
• Paper support medium contains carboxyl groups on
its surface
• Agarose, depending on the purity grade contains
sulfate groups
• The surface of glass walls used in capillary
electrophoresis contains silanol (Si-OH) groups

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 15
 ELECTROENDOSMOSIS

• Electroendosmosis (EEO) - movement of liquid

through the gel. This liquid originates from
• water used to prepare the gel, which indeed has to be
trapped by the gel material,
• movement of counter cations from both the gel and
buffer toward the cathode, while anionic groups in the
gel are affixed to the matrix and cannot move, gives rise
to EEO.
• Since electrophoretic movement of biopolymers is
usually toward the anode, EEO can disrupt
separations because of internal convection.

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 16
 Fig. 1. Electroosmotic flow through a glass capillary. electrolyte
cations are attracted to the capillary walls, forming an electrical
double layer. When voltage is applied, the net movement of
electrolyte solution toward the cathode is known as endosmotic
flow

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 17
 ELECTROPHORESIS AND
ELECTROENDOSMOSIS

• Silanol groups on the surface of glass ionize above pH 3

generating negatively charged sites
• It is these charges that generate electroendosomosis. The
ionized silanol groups create an electrical double layer, or a
region of charge separation at the capillary wall/electrolyte
interface. When voltage is applied, cations in the electrolyte
near the capillary wall migrate towards the cathode, pulling
electrolyte solution with them. This creates a net
electroosmotic flow toward the cathode.
• It is a diffusion process which is undesirable as it affects the
performance of electrophoresis

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 18
 SUPPORT MEDIA

• The pioneering work on electrophoresis by Arne Tiselius et

al. (1930) was performed in free solution
• It was soon realized that many of the problems associated
with this approach, particularly the adverse effects of
diffusion and convective currents could be minimized by
stabilizing the medium
• Stabilization was provided by use of a porous mechanical
support
• The support medium cuts down convective currents and
diffusion so that the separated components remain as sharp
zones

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 19
 SUPPORT MEDIA

• The earliest support materials were filter paper or cellulose acetate

strips, wetted in electrophoresis buffer. Not in common use in
recent times, though cellulose acetate still used
• For many years, small molecules like amino acids, peptides and
carbohydrates were separated and analyzed on supports such as
paper or thin-layer plates of cellulose, silica or alumina, but
presently analyzed by more sensitive methods such as high
performance liquid chromatography (HPLC)
• Separation of macromolecules such as proteins and nucleic acids
on paper or thin-layer plates was found to be poor
• Gels were therefore developed as support media for
macromolecules.

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 20
 THE GEL

• The earliest gel system used was starch gel. Today majority of
electrophoretic techniques involve agarose or polyacrylamide gels
as support media
• In most cases, the gel is a crosslinked polymer whose composition
and porosity is chosen based on the specific weight and
composition of the molecules being separated

• When separating proteins, small nucleic acids or

oligonucleotides, different concentrations of acrylamide and a
cross-linker molecule are used to produce varying- sized mesh of
networks of polyacrylamide
• When separating larger nucleic acids, usually greater than a few
100 bases the preferred matrix is purified agarose

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 21
AGAROSE GELS

• Agarose is a linear
polysaccharide (average Seaweed
molecular mass = 12,000
Daltons), made up of the basic
repeating unit agarobiose
• Agarobiose comprises
alternating units of 1,3-linked Gelling Temp: >35° C
β-D-galactose and 1,4-linked ► Formula:
(C12H14O5(OH)4)n
3,6-anhydro-α-L-galactose; ► Soluble in Hot Water
may have carboxylate, ► 100 g costs $307.75
pyruvate, or sulfate ► Manufacturers
substitutions. prepare special grades
of agarose for scientific
• Agarose is one of the experimentation
components of agar, a mixture ► Purified agarose is in
of polysaccharides isolated powdered form, and is
insoluble in water (or
from certain seaweeds buffer) at room
temperature

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 22
AGAROBIOSE - THE REPEATING UNIT
OF AGAROSE

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 23
 PREPARATION OF AGAROSE GELS

• Agarose is usually used at concentrations of between 1% and 3%.

For large DNA fragments (5–10kb) 0.7% gels give good
resolution. For small 0.2–1kb fragments 2% gels give good
resolution. Beyond 3%, gel become brittle and breaks easily
• Agarose dissolves in boiling water. In solution it forms a random
coiled structure.
• When it starts to cool, the agarose chains form helical fiber
bundles held together by hydrogen bonding. The the long agarose
chains crosslink with each other in junction zones by formation of
more hydrogen bonds, causing the solution to "gel" into a semi-
solid matrix

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 24
 PREPARATION OF AGAROSE GELS

• Increase in agarose concentration produces a firmer

gel
• While the solution is still hot, we pour it into a mold
called a "casting tray" so it will assume the shape of
the tray
• To make 40 ml of 1% agarose: weigh 0.4 g of
agarose, dissolve in 40 ml TAE buffer (Tris-Acetate-
EDTA). Pour on gel plate and allow to cool to room
temperature to form a rigid gel

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 25
 PREPARATION OF AGAROSE GELS
• Gelling properties of
agarose attributed to both
inter- and intramolecular
hydrogen bonding within
and between the long
agarose chains: cross-
linkages
• This cross-linked
structure gives the gel
good anticonventional
properties
Casting tray

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 26
 AGAROSE GELS

• Pore size of the gel is controlled by the initial concentration of

agarose
• Low concentrations form large pore sizes while high concentrations
form small pore sizes
• Also present on the agarose are substitutions of alternating sugar
residues with carboxyl, methoxyl, pyruvate and sulfate groups to
varying degrees
• This substitution can result in electroendosmosis during
electrophoresis: this is undesirable
• Also ionic interactions between gel and sample may occur: unwanted
• Agarose is therefore sold in different purity grades, based on the
sulfate concentration
• The lower the sulfate content, the higher the purity

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 27
 AGAROSE GELS

• Agarose gels are used for electrophoresis of both proteins and

nucleic acids
• For proteins, pore sizes of a 1% agarose gel are large relative to the
sizes of proteins
• Agarose gels are therefore used in techniques such as
immunoelectrophoresis or flat-bed isoelectric focusing, where the
proteins are required to move unhindered in the gel matrix
according to their native charge
• Such large pores of agarose gels are used to separate large
molecules such as DNA and RNA because the pore sizes in the gel
are large enough for these molecules to pass through the gel
• If molecular size increases, frictional effects begin to play a role in
the separation of these molecules

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 28
 AGAROSE GELS

• Advantage of using agarose is the availability of low melting

temperature agarose (62-65°C) gels which can be reliquefied
by heating to 65°C. After separation of the samples, DNA
bands can be cut out from the gel, returned to solution and
recovered
• Horizontal slab gels which make use of agarose are used for
immunoelectrophoresis and isoelectric focusing, as well as
routine separation of DNA and RNA

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 29
 OTHER SUPPORT MEDIA FOR GEL
ELECTROPHORESIS
• Polyacrylamide, starch
• Polyacrylamide gels: most commonly used gel for the following reasons:
• i) They are very stable
• ii) They can be prepared in a wide range of concentrations to give a
variety of pore sizes
• iii) They can be made to have a gradient of concentrations in one gel slab
• iv) Polyacrylamide gels good for separating proteins of size 5 to 2,000
kDa because unlike agarose it gives uniform pore size even at high
concentration

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 30
 OTHER SUPPORT MEDIA FOR GEL
ELECTROPHORESIS
• Polyacrylamide gel was widely used in the Maxam-Gilbert or
Sanger DNA sequencing for separation of small fragments up
to 1 bp difference

• Electrophoresis in acrylamide gels is often referred to as

PAGE (polyacrylamide gel electrophoresis)
Formation of polyacrylamide gel
• Two methods are used to polymerize acrylamide: chemical
polymerization and photopolymerization

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 31
 POLYACRYLAMIDE GELS
Chemical polymerization
You need the following reagents:
• Acrylamide as the monomer
• A cross-linking agent: N,N’-methylene bis-
acrylamide (normally referred to as bis-
acrylamide)
• An initiator of the polymerization process:
ammonium persulfate
• A catalyst: N,N,N’,N’-tetramethylenediamine
(TEMED)

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 32
 Reagents for chemical polymerization
of acrylamide

Acrylamide: molecular formula N,N,N’ N’-

C3H5NO tetramethylenediamine
Molar mass: 71.08 g/mol. A white (TEMED)- the catalyst
odorless crystalline solid. A known
neurotoxin and suspected
carcinogen

(NH4)2S2O8

Ammonium persulfate: molar mass 228.2 N,N’-methylene-bis-acrylamide:

g/mol. Free radical is produced in this molecular formula C7H10N2O2
way: Molar mass 154.17 g/mol
S2O82- + initiator catalyst → SO4 •‾+ SO4•‾ A crosslinking reagent

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 33
 CHEMICAL POLYMERIZATION

• Proceeds by a free radical polymerization process. Upon

addition of ammonium persulfate to acrylamide, the
ammonium persulfate dissolves in the water (used to prepare
the acrylamide reagent) to form free radicals This process is
short-lived, slow, hence needs a catalyst
• An additional catalyst/initiator TEMED is added to enhance
the free radical production from persulfate and continue the
polymerization process
• TEMED catalyzes the decomposition of persulfate ion to give
a free radical
• S2O82- + initiator => SO4 •‾+ SO4•‾
• The free radical initiates polymerization process through vinyl
addition of the monomers
Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 34
 CHEMICAL POLYMERIZATION

• Vinyl addition of acrylamide monomers occurs in a head-to-

tail fashion to build long and linear chains of
polyacrylamide
• Addition of bis-acrylamide leads to copolymerization of the
straight polymer chains to produce a cross-linked
polyacrylamide
• Bis-acrylamide is essentially two acrylamide molecules
linked by a methylene group and therefore is able to set up a
copolymerization reaction in which long and linear
polyacrylamide chains are occasionally interspersed with bis-
acrylamide molecule, thereby introducing a second site for
chain extension

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 35
Head-to-tail and copolymerization of acrylamide to
form a polyacrylamide network (gel) held together by
bis-acrylamide crosslinks

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 36
 FORMATION OF POLYACRYLAMIDE GEL
FROM ACRYLAMIDE AND BIS-ACRYLAMIDE

• The result is a fairly well-defined three dimensional network

of crosslinked polyacrylamide having a range of pore sizes
and rigidity
• The pore size and rigidity can be controlled by the
experimenter to enable separation of a wide range of sample
sizes.
• Pore size is controlled by changing the concentration and
ratio of acrylamide monomer and bis-acrylamide

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 37
 RATE OF POLYMERIZATION OF
ACRYLAMIDE GELS
• The rate of polymerization depends on
(1)The net concentration of monomers and initiators
(2)Temperature
(3)Purity of the reagents

All three should be controlled for reproducibility. Reagents should be

electrophoresis grade and deionized water should be used
• For highest quality resolution, dissolved oxygen should be removed from
the monomer mixtures by degassing them, since oxygen decreases the rate
of polymerization. This is done by briefly placing it under vacuum to
remove dissolved air prior to use. Degassing of the gel also serves to
remove heat that is liberated during polymerization.
• Polymerization of acrylamide is an exothermic reaction. Warming up of
the gel solution as it sets can liberate air bubbles that become trapped in
the polymerized gel.
• Air bubbles in the gel affect resolution of the sample
Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 38
 POLYACRYLAMIDE GELS: PORE SIZE
AND RIGIDITY

• The ratio of bisacrylamide to acrylamide, as well as the total

concentration of both components affect the pore size and rigidity of the
final gel matrix. These, in turn, affect the range of protein sizes
(molecular weights) that can be resolved

• By convention, polyacrylamide gels are characterized by a pair of values,

%T and %C, where %T is the concentration of total monomer (acrylamide
+ bis) in g/100 ml and %C is the concentration of bis alone as a
percentage of total monomer.

• The effective pore size of a polyacrylamide gel is an inverse function of

the total monomer concentration (%T) and a biphasic function of %C.
When %T is increased at a fixed %C, the number of chains increases and
the pore size decreases continuously. On the other hand, when %T is held
constant and %C is increased from low values, pore size decreases to a
minimum at about 5%C.
Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 39
 Pore
size

At 5% Bis-
concentration, pore Bis concentration
size is minimum

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 40
POLYACRYLAMIDE GELS: PORE SIZE
AND RIGIDITY

• With further increase in %C from the minimum of 5%, pore

size increases, probably due to the formation of shorter,
thicker bundles of polymer chains.
• A plot of pore size against %C should give a parabola
• Typically 5-20% by weight (5%, 7.5%, 10%, 12.5%, 15%,
20% are commonly used values). To vary pore size, fix
concentration of BIS at 5% and vary monomer concentration.
Implies gel is mostly water.
• Gels with low %T (e.g., 7.5%T) are used to separate large
proteins, while gels with high %T (e.g., 15%T) are used with
small proteins.

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 41
 PROPERTIES OF POLYACRYLAMIDE
GELS
• Polyacrylamide gels are hydrophilic and electrically neutral
at the time they are cast. They are transparent to light at
wavelengths above 250 nm and do not bind to protein stains.
• The gel has both solid (rigid fibers) and entrapped liquid
components which create a three-dimensional shape for the
gel. Without the liquid, the gel will dry to a thin film. At the
same time, the polymer fibers hold the liquid firmly and
prevent it from escape.
• Polyacrylamide gels are well suited for protein
electrophoresis

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 42
 POLYACRYLAMIDE GELS

• TEMED is a base, hence polymerization is most efficient at

alkaline pH. Polymerization efficiency falls rapidly at pH
values below 6
• Photopolymerization with riboflavin is used for low-pH gels

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 43
 POLYACRYLAMIDE GELS

Photopolymerization
• In this method, riboflavin replaces ammonium persulfate and
TEMED. When the gel is poured, it is placed in front of
bright light for 2-3 hours
• Riboflavin is decomposed by light to generate free radicals
that initiate polymerization of acrylamide
RE-cap on pore size of acrylamide gels
• Polyacrylamide gels can be made to have varying pore sizes
• The size of the pores within the gel can be varied/controlled
by changing the concentrations of both the acrylamide and
bis-acrylamide

Dr. Alexander Kwarteng || Department of Biochemistry & Biotechnology, College of Science, KNUST 44