Electrophoresis 247

Figure 10.8 Starch gel electrophoretic pattem of acid phosphatase

Agar Gel Electrophoresis

Agar gel has been much utilized for immunoelectrophoresis by
Graber and Williams (1959), Generally, a horizontal tank is used
for agar gel electrophoresis. To prepare agar gel (1-2%) on
microscopic slides, the agar solution is poured into a petri dish
first. It is allowed to cool and a flat surface is obtained. Microscopic
slides are placed on the gel and fresh agar solution about 2-mm
thick is poured again. The resulting gel is ofuniform thickness and
sample is loaded in circular wells made in the gel (Figure 10.9).
Buffer Light petroleum Buffer

Agar gel slide

Agar gel Agar gel

Figure 10.9 Agargel electrophoresis

Agarose Gel Electrophoresis

Agarose is a natural product purified from red seaweed
(Rhodophyta). It is a polysaccharide of alternate 1,4-linked 3,
6-anhydro--1-galactopyranose and 1,4-linked oa-b-galactopyranose
residue and arranged intó a double helix. It dissolves on boiling
and forms a gel when cooled.
In this clectrophoresis technique, agarose is used at a
concentration of 1 to 3%. Agarose gels are prepared by suspending
dry agarose in aqueous buller and then boiling the mixture until a
248 Bioinstrumentation

clear solution is formed, It is allowed to cool to

room temperature
to form a rigid gel. Numerous inter- and
bonds within and between the long agarose intramolecular hydrogen
chain molecules are
responsible for the gelling properties. This cross-linking gives the
gel good anticonvectional properties. The pore size is controlled by
the initial concentration of agarose. Low concentrations of
agarose
produce large pore size and high concentrationsproduce smaller
pore size.
DNA molecules having high molecular weights cannot
penetrate through even a weak cross-linked polyacrylamide gel.
This problem is solved by agarose. A 0.8% agarose gel can accept
DNA molecules with molecular weight as high as 59 × 10°. Gels
of different concentrations must be used according to the molecular
weight of DNA samples. This is necessary because very large
molecules cannot penetrate through the more concentrated gels
and very small molecules will pass through the dilute gels easily.
agarose gel electrophoresis technique, the glass slides on
which agarose gels are prepared are kept on the bridges of the
apparatus. The gel is connected to the anode and cathode buffer
reservoirs through paper wicks (Figure10.10). The sample is applied
in the wells made in the gel. When electrophoresis is complete, the
DNA is identified by immersing the gel in a dilute solution of the
fluorescent dye, ethidium bromide. After some time, the gel is washed
to remove unbound dye from the agarose and then it is illuminated
with ultraviolet light to excite the fluorescence of ethidium
Sample
Bridge
Lid

Paper wick
Gel slide Electrode
Buffer

Flgure 10.10 Agarose gel electrophoresis

 Electrophoresis 249

bromide. The gel with its fluorescent bands is then photographed.

Agarose gel can be usedfor immunoelectrophoresis technique.
Submarine Gel Electrophoresis
The most convenient method to study DNA and RNA fragments by
electrophoresis is by agarose gels (Sharp et al., 1973). Agarose gel
of desired thickness can be cast in specially made trays called 'gel
platform'. In order to make sample wells in gel, a suitable comb is
placed in the gel platform during gel casting. After the gel is set,
the comb is taken and rectangular wells are obtained. Agarose gel
along with the gel platform is placed in the appafatus horizontally.
Buffer is poured to submerge the gel and electrophoresis is carried
out. This method of electrophoresing the gel under submerged
condition is popularly known as submarine gel electrophoresis
(Figure 10.11).
Sample wel Gel platorm
Lid

Butfer

Gel

Figure 10.11 Submarine gel electrophoresis

After the electrophoresis, tlhe gel is stained with ethidium
bromide for detecting nucleic acids(Ethidium bromide binds with
DNA/RNA and gives fluorescent bands when viewed in the
(ransilluminator. Generally, Perspex sheets used to make gel
platforms are opaque. So the gel to be viewed should be taken out
of the gel platform carefully. This is very
the gel breaks while handling. Also, since difficult and sometimes
carcinogenic, it should be handled cautiously.ethidium bromide is
To overcome these
difficulties, gel platforms or the floor of the apparatus are made of
UV-transparent Perspcx sheet. In such cases bands can be viewed
Without taking the gel out and thus tthe handling process has become
very convenient and safe with the UV-transparent gel platlorms.
254 Bioinstrumentation

2. Relatively thin gels can be used in vertical gel which are

easily and quickly processed.
3. No extra pair of electrodes is needed.
4. By choosing an appropriate ramp (decrease in the pulse
rate during the course of a run) molecules can be resolved
within a limited size range. This sort of trick is very
essentially required in techniques like Restriction
Fragment Length Polymorphism (RFLP) analysis where
separation and accurate mneasurement of fragment must
be made.
5. FIGE is faster than other pulse field techniques: for example,
by using FIGE, DNA fragments between 10-15 kb can be
separated in 6 hours. In other pulse field techniques 5-10 kb
fragments take about few days for separation.
Applications
Agarose gel electrophorcsis is used to
1. isolate a large number of proteins
2. identify the purity of isolated proteins
3. determine the sequences of DNA
4. find out the presence of mutation in DNA or RNA
and northern blotting) (Southern
5. detect the precursor molecules cf tRNA, rRNA and
mRNA
6. study the kinetics of the interconversions of
in many tRNAS conformation
7. find out thenumber of subunits present in a protein
8. determine the molecular weight of
proteins and DNA
Folyacrylamide Gel Electrophoresis
Electtophoresis in acrylamide gel is frequently referred to as PAGE
(Polyacrylamide Gel Electrophoresis). The components used in PAGE
are acrylamide. bisacrylamide and TEMED (Tetramethyl ethyene
 Electrophoresis 255

diamine)Cross-linked polyacrylamide gels are formed from the

polymerization of acrylamide monomer in.the.presence of small
åmounts otbisacrylamide, The pore size in the gel can be varied by
changing the concentration of both the acrylamide and
bisacrylamide.
Several proteins of biological.importance contain more than
one polypeptide chain, These proteins are referred to.as oligomeric
proteinis. The structure of these proteins is stabilized by hydrogen
bonding, disulphide linkages or by hydrophobic interactionsiThe
subunits of these proteins_can be_separaced irom each other bya
class of conpounds known as solubilizers, e.g. urea, beta
mercaptoethanol and sodiun1 dodeyÍ sutptate (SDS).)
(Beta-mercaptoethangl breaks disulphide bridges present in the
oligomeric proteins SDslis an aniónic detergent) and disrpia
macromolecules whose structure has been stabilized by hydrophobic
interactions\,SDS binding imparts a large negative charge to the
denatured polypeptides
PAGE COmbined with.SDS is the most widely used method for
analysing protein mixtures quantitatively. It is particularly usefür
for monitortng protein puriliicaion, In 1967, A. Shapiro.
£ Vinuela and J. Maizel showed that the molecular-weight of
most proteins could be determined,by.measuring the mobility of
most proteins in polyacrylamide gels containing SDS)
Initially, the sampleto be separated is boiled for five minutes
in a sample buffer containing SDS and beta-mercaptoethanol.This
Lreatment completely denatures proteinspresent inthe sample and
imparts negative charge tothe polypeptide chains. The sample buff'er
álso contains an tonizable tracking dye (c.g.
which allows to monitor the ciectrophoretic run.)bromophenol blue).
sps-PAGE Is usually carried out as. column elecuophoresis
(Figure 10.15). The gelis polynerized tn a column. This column is
then fitted between the upper and lower bufer reservoiIs. An
electrophoretic apparatus usually contains 8to-12 colunns.
256 Bioinstrumentation

Butter
Sample
Stacking gel
Separating gel.

Butfer

Figure 10.15 SDS-PAGE

In SDS-PAGE, the gel consists of two parts, namely, the main
separating gel (about10cm long) anda shorter stacking gel (about
1cm long). The main separating gel is first poured into aglass
tube andallowed to set. Then 1 mlofstacking gel is pouredon the
top ofthe separating gel. The stacking gel has a verylarge pore size
gel has comparatively small pore size,
and the separating
Samples of proteinsof known andunknown molecular weight
are layered on the top of each column
separately. The stacking gel
allows the proteins to move freely and çoncentrate over the
separating gel under the influence of the electrc held, Proteins
Coninue their movement towards the anode. Since proteins have
thà same charge per unit length, all proteins travel with the same
mobility. However, as they pass through the separating gel, the
proteins separate owing to the molecular sieving PIoperties ofthe
gel. The smaller proteins mave fast as rhey carn pass thfough the
pores of the gel. But large protcins move slowly since they are
retarded by trictional resistance due to sieving ettects of gel. When
the dyereaches the botom ofthe gel, th cårrentis turned olt. The
gei is removed ftom the glass tube and stained with appropriate
Stain solion (Figure 10.16).
A plot of the distance migrated versus log of the molecular
weight gives a straight line. Hence, if a protein ofunknown
 Electrophoresis 257

molecular weight is electrophóresed with two.or more of proteins of

known mnolecular weight, the molecular weight of unknown protein
can be calculated to an accuracy ranging between 90 and 95 per
cent (Figure 10.17). This is the most common way of estimating
the molecular weight of protein subunits.

SDS
Polyacrylarnida gel

Subunits

Figure 10.16 Separation of enzyme subunits by SDS-PAGE

(x10")

w.
mol.
Log

1 2
Distance migrated (cm)
Figure 10.17 Determination of molecular weight
ISOELECTRIC FOCUSING
The method of scparating proteins according to their isoelectrie
Ponts inaplB gradient is catted 1soelectric fecusing. This technique
Was discovered by H. Svensson in Sweden. This method has a
258 Bioinstrumentation

high-resolution power because ordinary paper electrophoresis

resolves plasma proteins into six bands whereas isoelectric focusing
resolves them into 40 bands)
An conventional electrophoresis, the pH between anode and
cathode is constant and the positively charged ions migrate
towards the cathode and negatively charged jons migrate towards
theanode. But in isoelectric_ focusing,a stable pH gradient is
aranged. The pi gradually increases irom anodetocathode. When
a protein is introduced at a. pH which is lower than its isoionlc
point, it will possess a net positive charge and will migrate in the
direction of the cathode. Due to the presence of pH gradient, the
net charge of the molecule changes due to ionization as it moves
forward. When the protein encounters a pH where its net charge
is zero, it will stop migrating,/This is the isoelectri point of protein,
Each protein present in the mixture will migrate to its isoelectric
point and stop its migration at that point (Figure 10.18))Thus,
once a final, stable focusing is reached, the resolution will be
retained for a long time.
Direction of migration
of anions Direction of migration
of cations
Zwitterions

Cathode
Anode
w
Xy

High pH
Migrating Low pH
sample Migrating
sample
components components
(anions)
(cations)
Sample Introduced
at pH above
Immoblzed sample
components at their Sample introducgd
Isoelectric polnt isoelectric points at pH below
Isoelectric point
Figure 10.18 Isoelectric focusing
()Isoelectric focusing is widely, used for.the separation and
identification of serum proteins/t 0s used in thefood and agriculture