Block 1 Techniques in Cell Biology

UNIT 3
TECHNIQUES IN
MOLECULAR BIOLOGY

Structure
3.1 Introduction Differential centrifugation
Objectives Ultracentrifugation
3.2 Spectrophotometry 3.4 X-ray Diffraction
Principle of 3.5 Electrophoresis
spectrophotometer
3.6 Summary
Working and applications
3.7 Terminal questions
of spectrophotometer
3.8 Answers
3.3 Centrifugation
Density gradient
centrifugation

3.1 INTRODUCTION
In the previous unit you have studied about the chromatography techniques
used for the detection and separation of some biological compounds. This unit
will introduce you to some other techniques used in cell and molecular biology.
These techniques also help in the separation of various molecules,
compounds and cellular structures such as cell organelles. The components
separated using these techniques increase our knowledge about structural
details and various cellular processes associated with them. The present unit
provides detailed information about spectrophotometry, centrifugation, X-ray
diffraction analysis and electrophoresis techniques.

Objectives
Objectives
After studying this unit, you would be able to :

describe the principles and application of spectrophotometry;

describe the technique of centrifugation;

enlist different types of centrifugation;

describe the technique of X-ray diffraction analysis; and

58
provide a detailed information about electrophoresis technique.

Unit 3 Techniques in Molecular Biology

3.2 SPECTROPHOTOMETRY
Spectrophotometry is a technique that measures the quantity of compounds
present in any solution. It is the branch of spectroscopy that measures
electromagnetic radiation absorbed, transmitted or reflected by any chemical
substance over a certain range of wavelength. The technique of
spectrophotometry is used for quantitative analysis of various chemical
compounds. Spectrophotometer is the instrument used for this purpose. The
concentration of a substance present in a solution can be quantified by
measuring light absorbed by it at a particular wavelength. Spectrophotometers
have been classified into two types on the basis of range of wavelength of light
source used in the instrument:

1) UV- Visible Spectrophotometer: In this type of spectrophotometer

absorbance can be measured in the UV (ultraviolet) range (185 to
400nm) and visible region of the electromagnetic spectrum (400 to
700nm).

2) IR –Spectrophotometer: This type of spectrophotometer measures

absorbance in the infrared range (700 to 1500nm) of electromagnetic
spectrum.

3.2.1 Principle of Spectrophotometer

Working of a spectrophotometer is based on the photometric technique. When
a beam of light is passed through the coloured solution, some amount of light
gets absorbed and rest gets transmitted. A coloured solution absorbs all
colours of white light but transmits its own colour. Spectrophotometer
measures the amount of light that a sample absorbs when a beam of light is
passed through it. A beam of incident light of intensity (I0) passes through a
solution, a part of the incident light is reflected (Ir), a part is absorbed (Ia) and
rest of the light is transmitted (It). The spectrophotometer works on the
principle of Beer-Lambert’s law. This law states that the amount of light
absorbed by a colored solution is directly proportional to the concentration of
the solution. It is represented by the equation

A ∝ cl where,

A = Absorbance / Optical density of solution

c = Concentration of solution

l = Path length

or A = ∈cl

∈ = Absorption coefficient

Spectrophotometer helps in the quantitative measurement of coloured

compound present in the sample.

3.2.2 Working and Applications of Spectrophotometer

A spectrophotometer consists of a light source, a monochromator which is a
wavelength selection device, filter that produces rays of single wavelength, a 59
Block 1 Techniques in Cell Biology
sample chamber, a light detector, a display device, and computer storage set
up (Fig. 3.1).
3.1). Spectrophotometer can be single beam or double beam. Single
beam spectrophotometer uses a single beam of light and operates between
325 nm to 1000 nm wavelength. In this type of instrument, light travels in one
direction and same light source is used for reading blank and samples. Double
beam spectrophotometer operates between 185 nm to 1000 nm wavelength. It
has two photocells. This instrument splits the light from the monochromator
into two beams. One beam is used for reference and the other for sample
reading. It gives more accurate and precise results because the errors
occurring due to fluctuations in the light output and the sensitivity of the
detector get eliminated in the process.

Fig. 3.1: Photograph of a spectrophotometer.

The instrument is calibrated using the standard solutions. The calibration is

done with the known concentration of the solute whose quantity needs to be
determined in the test solution. The standard solution is filled in the cuvette
and placed in the cuvette holder present in the spectrophotometer. A ray of
light of certain wavelength (specific for the assay) is directed towards the
solution. The ray of light passes through a prism. The prism splits the beam of
light into different wavelengths. When the monochromatic light (light of one
wavelength) reaches the cuvette containing the standard or test solution,
some amount of light is absorbed by the solution and the remaining is
transmitted through the solution. The photodetector system measures the
intensity of transmitted light and converts it into the electrical signals. The
electrical signals are displayed
displayed in the digital form. The digits represent the
absorbance or optical density of the solution analyzed. If the absorption of the
solution is higher than more light will be absorbed by the solution (Fig. 3.2).

The absorption spectrum varies from UV (ultra-violet) or visible region of the

electromagnetic spectrum depending upon the compound quantified.

60 Fig. 3.2: Diagram showing basic set up of a spectrophotometer.

Unit 3 Techniques in Molecular Biology
Applications

The spectrophotometer is commonly used for determining the concentration of

coloured as well as colourless compounds via measurement of optical density
or absorbance. It can also be used for determining the course of the reaction
by measuring rate of formation and disappearance of the compound in the
visible and UV region of electromagnetic spectrum. Spectrophotometer is also
used in various industries like paints, plastics, paper, metals and fabrics.

SAQ 1
a) State the law on the basis of which spectrophotometer works.

b) Define spectrophotometry. Draw a neat and labelled diagram showing

the basic setup of a spectrophotometer.

3.3 CENTRIFUGATION
Centrifugation is a technique that is based on the use of the centrifugal force
to separate particles from a solution according to their size, shape, and
density. The principle behind the separation of components from suspension is
the centrifugal force/acceleration. The acceleration/force causes the denser
molecules to move towards the periphery while the less dense particles move
to the centre.

This technique involves various steps. The samples are homogenized and the
suspension is placed in a centrifuge tube. The tube is then placed in a rotor
and spun at a defined speed (Fig. 3.3). The rotation at a fixed point (axis),
result in strong force perpendicular to the axis. The perpendicular force
created during the rotation helps in the separation of components. The rate of
centrifugation depends on size and density of the particles present in the
solution. The components move with different speeds depending upon their
size and form series of bands. In a homogenate suspension, the cell
components are separated on the basis of their size.

Fig. 3.3: Photographs of benchtop centrifuge and refrigerated high speed

centrifuge. 61
Block 1 Techniques in Cell Biology
Relative centrifugal force (RCF or g) is the force acting perpendicular on the
sample relative to the gravity of the earth. It is the force exerted on the
contents of the rotor as a result of the rotation. The formula to calculate the
relative centrifugal force (RCF/g) is given as:

RCF (g) = 1.118 × 10-5 × r × (rpm)2

where r is the radius of the rotor (in centimeters), and rpm (revolution per
minute) is the speed of the rotor.

The technique helps in separation of various cell components.

3.3.1 Density Gradient Centrifugation

In this type of centrifugation, the molecules present in a heterogeneous
rpm/g can be
mixture settle down or get sedimented under a centrifugal force or under the
converted into RCF
by the following influence of gravity. The cell components or contents of sample get separated
formula. as distinct bands. The distribution of contents in different layers depends upon
the density. Larger and denser particles sediment faster in comparison to
rpm = [ RCF/r ×
small, less dense particles. A sucrose solution is used in the process. The
5
1.118)] × 1 × 10 centrifuge tube is filled with the gradient of sucrose and sample solution is
layered on the top of the column. The density of the solution increases
towards the bottom of the centrifuge tube (Fig. 3.4). The rate of sedimentation
depends upon the size of components. This is known as sedimentation
coefficient (Svedberg units). The Svedberg unit (S) is a measure of the
sedimentation rate of a particle when centrifuged. It is a measure of time and
is equal to the value of 100 femto seconds (10-13 s).

Fig. 3.4: Diagrammatic representation of various steps involved in density

gradient centrifugation.

The molecules such as DNA and RNA get separated by this technique. The
speed of 80,000 rpm produces a force equivalent to 500,000 times of gravity
which helps in the separation of small molecules (such as tRNA). Various
cellular activities such as synthesis of membrane bound proteins, formation of
transport vesicles, DNA synthesis and transport of solutes etc can be studied
using this technique. The technique can be applied for the purification of
62 biomolecules and separation of cell organelles.
Unit 3 Techniques in Molecular Biology
3.3.2 Differential Centrifugation
In this type of centrifugation, components get separated under the influence of
Centrifugal force
increasing centrifugal force. The basic principle of the technique is that rate of
sedimentation of biological particles differs due to difference in size and An outward force
density. The sedimentation of the larger molecules occurs under the influence experienced by an
of high centrifugal force. The sedimentation of smaller molecules takes longer object moving in a
time and requires much higher force. The large particles form a pellet on the circular path directed
bottom of the centrifuge tube, while small particles remain in the supernatant. away from the center
of rotation.
The settling of the particles depends upon the speed, time of centrifugation,
density and relative size of the particles.

Fig. 3.5: Various steps in differential centrifugation. 63

Block 1 Techniques in Cell Biology
The sample is homogenized in the medium containing buffer. The
homogenate is placed in the centrifuge tube and subjected to a centrifugal
force for a specific time at a particular temperature. The pellet formed at the
bottom of the tube is separated from the supernatant. The supernatant is then
added to a new centrifuge tube and subjected to another speed for a particular
time and particular temperature. The supernatant is separated from the pellet
formed again and again. The steps are repeated until all particles are
separated from each other.

This technique proves very useful for separation of cell organelles. Cells are
homogenized in an isotonic buffer solution (to prevent rupture of membrane
vesicles). The homogenate is subjected to a series of centrifugations
(sequential) with increase in centrifugal force. Initially the homogenate is
subjected to low centrifugal force for a short period of time. This separates the
larger cellular organelles such as nuclei. These organelles get sedimented as
a pellet. At higher centrifugal forces, larger cytoplasmic organelles such as
mitochondria, chloroplasts and lysosomes get extracted from the suspension.
In subsequent steps, microsomes (fragments of vacuoles and membranes of
the cytosol) and ribosomes are removed from suspension (Fig. 3.5). The
organelles get separated in different fractions viz. nuclear fraction,
mitochondrial fraction, microsomal fraction and soluble fraction. Ultra or high
centrifugation normally occurs at the speed of 75,000 revolutions per min and
produce forces equivalent to 500,000 times that of gravity.

3.3.3 Ultracentrifugation
Ultracentrifuges are the highly sophisticated and advanced type of centrifuges
that operate at extremely high speeds. These centrifuges are large in size and
separate samples in large batches and in a continuous flow system. They help
in separation of smaller molecules like ribosomes, proteins, and viruses. They
help in separation of molecules that cannot be separated with other
centrifuges. The speed of these centrifuges ranges from 60,000 to 150,000
rpm. Most of the ultracentrifuges are refrigerated because this helps in
balancing heat produced during intense spinning.

64 Fig. 3.6: An ultracentrifuge.

Unit 3 Techniques in Molecular Biology

The working of this centrifuge is based on the principle of sedimentation. The

denser particles settle down fast under the influence of gravity (Fig. 3.6). The
molecules get separated on the basis of their buoyant density. The band of
highest buoyant density is at the bottom while one with lowest buoyant density
is at the top. The separation is independent of size and shape. A high
concentration of sucrose or cesium chloride is used in this type of
centrifugation. Each component moves down and reaches a position where
the density of the solution is equal to its own buoyant density (Fig. 3.7).The
denser molecules move outwards to the periphery of the tube whereas the
less dense molecules get inclined towards the center of the tube.

Fig. 3.7: Various steps involved in ultracentrifugation.

Ultracentrifuges can also be used for the determination of properties such as

size, shape, and density of macromolecules. Ultracentrifuges are used for
separation of cell organelles like mitochondria, ribosomes and viruses. The
macromolecular conformational changes due to changes in pH, temperature,
and other environmental factors can be detected using this instrument. The
densities of various macromolecules can also be determined using this type of
centrifuge. The purification of various biological crude extracts can also be
made using this instrument.

Besides these, some centrifuges are also available that possess temperature
control system required for certain special processes. Refrigerated centrifuges
provide temperature control ranging from -20° C to -30° C. Microcentrifuges
are the centrifuges used for the separation of samples with smaller volumes
ranging from 0.5 to 2 µL. They usually operate at a speed of about 12,000-
13,000 rpm and are used for the molecular separation of cell organelles like
nuclei and DNA and phenol extraction. Microcentrifuges are also called
microfuges. 65
Block 1 Techniques in Cell Biology

SAQ 2
Fill in the blanks with appropriate words:

a) The spectrophotometer works on the principle of ………………….. .

b) In a spectrophotometer, the concentration of colored compounds is

measured as ………………… .

c) ……………….. gives the measure of the strength of rotors used during

centrifugation.

d) In differential centrifugation, components get separated by increasing

………………… .

e) The centrifuges used for the separation of samples with smaller volumes
are known as ……………………. .

3.4 X RAY DIFFRACTION

X-Ray Diffraction (XRD) is a technique used to analyze the structure of
crystalline materials. The technique is used for analyzing the atomic or
molecular structure of materials. The chemical composition of the crystalline
structures can also be known through this technique. The structure of
crystalline materials depends on the dual wave/particle nature of X-rays. The
atomic structures determined by X-ray crystallography indicate the role of
individual molecules that make up a multisubunit complex.

Most of the materials are composed of many tiny crystallites called as

polycrystalline aggregates or powder. The finely ground powder with
crystallites is placed in an X-ray beam for analysis. XRD reveals the geometry
or shape of a molecule. The scattering of X-rays from structures determine
the shape or geometry of the molecule. The X-rays get diffracted by a crystal
and provide the inter-atomic spacing in the crystals. When an incident beam of
monochromatic X-rays hit the three-dimensional arrangement of atoms in a
crystal, X-rays get scattered after striking the atoms within the target material
(Fig. 3.8).

66 Fig. 3.8: Diagram showing principle of X-ray diffraction.

Unit 3 Techniques in Molecular Biology
Most of the X-rays destructively interfere with each-other and cancel each-
other out, but in some specific directions X-ray beams interfere constructively
and reinforce one another. The reinforced X-rays produce the characteristic
diffraction pattern which helps in determining the structure of crystal.
Diffraction occurs when light bends slightly as it passes around the edge of an
object or encounters an obstacle or aperture (Fig. 3.9). The degree of
diffraction depends mainly on the size of the material. The direction of
diffraction depends on the size and shape of the material, while the intensity of
diffraction depends on the arrangement of atoms in the crystal structure.

Fig. 3.9: Representation of the setup for XRD technique.

The technique is based on the principle of W.L. Bragg which states that
diffracted X-rays act as rays reflected from various planes within the crystals.
These planes were later named as Bragg's planes and mainly include rows of
atoms that make up the crystal structure. The Bragg's equation is represented
as follows:

n (λ)= 2d Sinθ

where ‘n’ is an integer (1,2,3,...n),

λ (lambda) is the wavelength,

d is the distance between the atomic planes, and

θ (theta) is the angle of incidence of the X-ray beams.

The technique got advanced with the passage of time. Synchrotrons are being
used to generate highly focused and intense X ray beams. The beams
possess high energy particle accelerators. Highly sensitive electronic
detectors (charged coupled devices, CCD) that provide a digital readout of the
diffraction data are used.

The XRD technique is mainly used in the identification of unknown crystalline

materials. The method proves useful for studying minerals, polymers and
unknown materials. The tertiary structure of proteins and structure of large
molecular structures can be determined using this technique. The technique
has got applications in the field of Geology, Engineering, Biology, Material
science, Environmental science, and Pharmaceuticals.

3.5 ELECTROPHORESIS
Electrophoresis is a technique by which biomolecules are separated based on
their size and electrical charge. In this technique, the charged particles 67
Block 1 Techniques in Cell Biology
migrate under the influence of an electric field. The separation of the
molecules depends on their ability to move in an electric field. When placed in
an electric field, molecules get separated on the basis of difference in their net
charge. The technique was introduced by Swedish chemist Arne Tiselius in
1930.

Biomolecules are electrically charged particles and possess ionizable

functional groups. They exist as either positively or negatively charged ions in
a solution of a given pH. When subjected to an electric field, the ionized
biomolecules migrate depending on the mass and the net charge of each
particle. The negatively charged particles (anions) migrate towards a positively
charged electrode (cathode), while the positively charged particles (cations)
move towards a negatively charged electrode (anode).The differences in the
speed and the direction of each charged particle result in a unique migration
pattern leading to the isolation of components of the biomolecules that
possess similar characteristics.

Types of Electrophoresis

The electrophoresis can be of different types depending on the type of buffer

solution used and mobility of the charged particles. The major types of
electrophoresis include:

Moving boundary electrophoresis - In this type of electrophoresis, the

samples are present in solution with constant pH. The separation is low
because mixing of samples occurs in the buffer solution which results in the
overlapping of components or particles that possess similar characteristics.
The mobilization of separating particles can be measured without intervening
factors.

Zone electrophoresis (ZE) - Here the separation takes place in a

homogenous buffer system. Samples are surrounded with electrophoretic
buffer solution and separated in the matrix for a definite separation time. The
matrix provides an additional sieving effect for the separation. The matrix or
supporting medium controls diffusion of sample. The technique is widely used
in biochemistry and molecular biology research. It is also useful in biochemical
investigations.

Isotacho-electrophoresis or isotachophoresis (ITP) - It is a form of

electrophoresis in which ions migrate at same velocity. The samples are
placed between two non-homogeneous buffer solutions composed of leading
electrolyte at one electrode and a terminating electrolyte at the other end (Fig.
3.10). The leading electrolyte shows the highest mobility. Thereafter follow the
charged particles and terminating electrolytes under the influence of electric
current. This type of electrophoresis is carried out in capillaries. The
advantage of this method is that it does not require any sample preparation
and is applicable to wide range of samples.

Isoelectric focusing (IEF) - Isoelectric focusing or IEF is the process of

separating charged macromolecules like proteins or peptides. The separation
68 depends on their isoelectric point or the pH at which a particular molecule
Unit 3 Techniques in Molecular Biology
does not have any charge. This type of electrophoresis is performed in a pH
gradient, which runs from low to high or from anode to cathode. This technique
is applicable only to amphoteric molecules because they can donate and
receive protons acting as acid and base.

Terminating electrolyte

Injected sample

Leading electrolyte

Electrode
sss
Conductivity detector

Semipermeable membrane

Leading electrolyte

Fig. 3.10: Isotachophoresis (Diagrammatic representation).

Electrophoresis can be performed in both one- and two-dimensions (2D). In

two-dimensional electrophoresis, second electrophoretic separation is carried
out in a direction perpendicular to the first. 2D-electrophoresis has shown to
provide better resolution and has found its use in analysis of clinical and field
samples.

Gel Electrophoresis - In gel electrophoresis biological molecules get

separated on the basis of their size. The molecules get separated by placing
them in a gel with small pores and creating an electric field across the gel. The
gel acts like a permeable matrix/sieve through which molecules can travel
when electric current is applied. Smaller molecules migrate more quickly and
travel far through the gel particles under the influence of electric current. The
separation of molecules is based on their size and electric current.

The gel is cast into strips or slabs with slots or sample wells. It is immersed in
an electrophoresis buffer solution before complete polymerization. Each well
of the gel is loaded and electric current is applied. The components in the
samples get separated on the basis of their mass under the influence of
electric current. Gel electrophoresis is commonly used in research and routine
diagnosis. The separation of various biomolecules can be achieved by
changing the type of polymers used in casting a gel or altering the pore size of
the gel.

There are two types of gel electrophoresis: native and denaturing. 69

Block 1 Techniques in Cell Biology
Native gel electrophoresis - In this type of electrophoresis, RNA or protein is
maintained in its native structure during electrophoresis. This type of
electrophoresis is usually used to identify large protein complexes.

Denaturing gel electrophoresis – In this type of electrophoresis, RNA or

protein is reduced i.e., present in its linear structure. The denaturation of the
RNA or protein is done by adding a reducing agent to the sample, gel and/or
buffer. The reducing agent separates bonds within the RNA or protein
molecule thereby reducing its secondary structure. This type of
electrophoresis helps in knowing the size of the molecule.

Both polyacrylamide and agarose gels are used in electrophoresis.

Polyacrylamide is compound which forms transparent gel after co-
polymerization of acrylamide monomers. Along with it a cross linking agent
such as N, N-methylene-bisacrylamide (‘bis-acrylamide’) is used. The
polymerization reaction is catalyzed by ammonium persulphate (APS) and
N,N,N’,N’-tetramethylenediamine (TEMED). DNA and proteins are separated
using a low percentage of acrylamide gel (3%-15%). Acrylamide gel of about
10%-20% is used in sodium dodecyl sulfate -polyacrylamide gel
electrophoresis (SDS-PAGE) in which separation of proteins occurs in
denatured condition (separation according to their size).

Agarose is a natural polysaccharide made of galactose and 3, 6-

anhydrogalactose chains and is obtained from agar isolated from red
seaweeds. To cast a gel, agarose powder is dissolved in a buffer solution,
heated and allowed to cool to room temperature. The concentration of the
agarose in the buffer solution determines the pore size of the gel. Agarose gel
of about 0.8% (w/v) to 5% (w/v) is used to separate DNA and RNA molecules.

Pulsed-field Gel Electrophoresis (PFGE)

In pulsed-field gel electrophoresis (PFGE), two electrical fields are applied

periodically in rotation at different angles. High-molecular-weight DNA
molecules generally occur in a coiled state and get stretched after application
of electric field in a gel. Alteration in the direction of the electric field helps in
the reorientation of large DNA molecules. Two alternating electric fields from
different angles help in revealing migration distance for small quantity of high-
molecular-weight DNA molecule. Generally shorter migration distance has
been noted in case of large high-molecular-weight DNA. This type of
electrophoresis is used for the separation of chromosomes and high-
molecular-weight DNA molecules (more than 20 kb).

Capillary Electrophoresis (CE)

Capillary electrophoresis (CE) or High-Performance Capillary Electrophoresis

(HPCE) is performed in a narrow capillary immersed in an electrolyte buffer.
The capillary is typically 20-30 cm long and have inner diameter of 25-75 µm.
A sample is injected into the capillary which is now subjected to high voltage,
pressure and electric field. The sample components get separated along the
length of the capillary. The separated components are revealed by detector.
The major advantage of this technique is that a small sample volume can be
70 used for the separation.
Unit 3 Techniques in Molecular Biology
Immunoelectrophoresis
In this type of electrophoresis, antigens including proteins and peptides get
separated on the basis of their reaction and specificity to antibodies or
immunoglobulins (Ig). The binding of antigen to its corresponding antibody at a
specific point results in the precipitation of antigen-antibody complex. This type
of electrophoresis can be performed as a one-dimensional or two-dimensional
mode.

Affinity Electrophoresis
It is a type of electrophoresis in which separation of biomolecule is achieved
after interaction or binding to another molecule having a similar affinity. The
technique is based on the principle that electrical mobility (µ) changes when a
biomolecule including nucleic acids, proteins, peptides, and polysaccharides
bind to another molecule. The change in the electrical mobility gets reflected in
the electrophoretic pattern.

Set up for Electrophoresis

An electrophoresis apparatus consists of two opposite charged electrodes
(anode, cathode) connected by a medium called an electrolyte (conducting
solvent). In electrophoresis chamber, a power supply and an electrophoresis
unit are present together. The power supply provides the uninterrupted electric
current to the chamber. The chamber is composed of two electrodes, cathode
and anode, along with a reservoir containing buffer solution (Fig. 3.11).

Fig. 3.11: Set up for electrophoresis.

Gel casting trays, glass plates, and combs are other requirements essential for
gel casting. An internal cooling system is a prerequisite to provide thermo-
stable condition during electrophoresis and prevent excessive heating.
Components present in the sample separated after electrophoresis are not
visible, therefore detection and analytical methods are required for the
interpretation of results obtained.
The separated samples or components are generally not visible under normal
light, staining of gel is done. The staining varies for each type of molecule.
Coomassie Brilliant Blue or silver is used for staining proteins.

Ethidium bromide (dye) binds itself to nucleotide bases and fluoresces under
short-wave ultraviolet (UV) light (Fig. 3.12). The stained gel is visualized using 71
Block 1 Techniques in Cell Biology
a Gel Documentation System. The image of the gel can be procured through
this system as it is equipped with a camera. The gel image tells us about the
pattern of migration of bands (Fig. 3.13).

Fig. 3.12: An image showing gel in an electrophoresis chamber.

Besides staining, several other approaches such as autoradiography is also
used. In autoradiography, the molecules of interest are labeled with
radioisotopes.

Fig. 3.13: A photograph showing Gel Documentation System.

Principle of gel electrophoresis

The principle of gel electrophoresis is based on the fact that smaller molecules
move faster when present in the gel matrix in comparison to large molecules
that remain in the matrix. The small DNA molecules easily slip between the
various components of the gel matrix. The rate of migration of molecules
through the gel matrix depends upon the structure and charge of the proteins.
When proteins are separated by electrophoresis through a gel matrix, smaller
proteins migrate faster due to less resistance from the gel matrix. The use of
sodium dodecyl sulfate (SDS) or sodium lauryl sulfate and polyacrylamide gel
eliminates the
the influence of the structure and charge, and proteins get
separated only on the basis of length of polypeptide chain. SDS is a detergent
which denatures proteins after binding to its backbone. The binding of SDS
(reducing agent) cleaves disulfide bonds, help
help in unfolding of proteins into
72 linear chains.
Unit 3 Techniques in Molecular Biology
Procedure
Key points of the gel electrophoresis include:

• TAE (Tris acetate EDTA) buffer is used as a source of ions for setting up
the electric field during electrophoresis.

• The percentage of gel used depends upon the size of molecules. A high
percentage agarose gel (>1%) is prepared for separation of smaller size
molecules (<500bp). High percentage of agarose gel creates small
pores that increase the separation of small DNA segments.

• The agarose TAE buffer solution is poured into the casting tray. The gel
solution gets solidified after cooling. The wells are created at the top of
the gel using a comb.

• The solid gel is placed into a chamber filled with TAE buffer. The gel is
positioned in such a way that wells are placed close to the negative
electrode of the chamber. The wells are loaded with the DNA samples. A
DNA ladder or marker with multiple molecules of known and varying
molecular weights is also loaded as reference for size. The power supply
is switched on to generate electric field.

• The negatively charged DNA samples migrate through the gel and move
away from the negative electrode towards the positive one under the
influence of electric field. The rate of migration is proportional to size.
Smaller fragments move faster and reach bottom of the gel.

• Once the dye in the DNA samples migrates far enough through the gel,
the power supply is turned off and the gel is removed. It is then placed
into an ethidium bromide solution. Ethidium bromide intercalates
between DNA. Illumination with ultraviolet light causes the intercalated
dye to fluoresce.

• The stained gel is then exposed to UV light and a picture is taken. DNA
bands are visualized from each lane corresponding to a chamber well.
The DNA ladder gives us an estimation of the length of the DNA band.

Polyacrylamide gel electrophoresis (PAGE)

Polyacrylamide gel electrophoresis (PAGE) is an analytical method for
separation of components of a protein mixture based on their size. A
polyacrylamide gel is formed as a thin slab between the two glass plates. After
the gel gets polymerized, the slab is suspended between two compartments
containing buffer and two electrodes. The protein sample is prepared in a
solution containing sucrose or glycerol. Voltage is applied between the buffer
compartments. The alkaline buffers help in the separation as they make the
proteins negatively charged and cause them to migrate towards the positively
charged anode (see Fig. 3.12). The current flows across the slab causing the
proteins to move towards the oppositely charged electrode. The movement of
proteins depends on the charge density (charge per unit mass). The greater
the charge density, forcefully the protein molecule moves through the gel, and
high is rate of migration. The larger the protein molecule, more it becomes
entangled and slower it migrates. Compact globular proteins move more
rapidly in comparison to fibrous proteins. The lower is the concentration of 73
Block 1 Techniques in Cell Biology
acrylamide, less the cross linking in the gel and rapid is the protein migration.
A gel containing 5% acrylamide is generally used to separate proteins of 60 to
250kDa, whereas the gel of 15% is used to separate molecules of size 10 to
50 kDa.

A tracking dye is used to assess the progress of electrophoresis. The dye

Coomassie Brilliant Blue is commonly used to study proteins. The proteins can
also be radiolabelled. The position of proteins can be determined by pressing
the gel against
against the piece of X ray film. This results in production of an
autoradiograph. The gel is sliced into fractions and individual proteins are
isolated or gel is transferred by a second electrophoretic procedure to the
nitrocellulose membrane to form a blot. The proteins get absorbed on the
surface of the membrane.

Sodium dodecyl sulphate polyacrylamide gel electrophoresis

(SDS-PAGE)
Sodium dodecyl sulphate polyacrylamide gel electrophoresis is the most
commonly used gel electrophoresis. The technique facilitates better separation
and analysis of proteins. Sodium dodecyl sulphate (SDS) a negatively charged
detergent binds to large number of protein molecules. The polyacryalmide gel
forms an inert matrix through which proteins migrate in SDS-PAGE. The SDS
solubilized proteins when passed through the gel migrate towards the positive
terminal electrode. SDS binds to the hydrophobic regions of the protein
molecules and causes them to unfold into extended polypeptide chains.
reducing agent is also used which helps in breaking of S-S
Mercaptoethanol, a reducing
(disulphide) linkages so that protein subunits can be separated. Both SDS and
mercaptoethanol help in unfolding the native structure of protein.

The unfolding of proteins into a rod shape eliminates

eliminates the differences in shape
of proteins. This helps in proteins of same size to behave similarly and
proteins of different sizes form series of discrete bands. The number of SDS
molecules that can bind to protein is roughly proportional to the molecular
mass of the protein. The proteins get separated on the basis of molecular
mass i.e. polypeptides are separated due to size. The gel can be stained with
dye Coomassie Brilliant blue to visualize major bands while the minor bands
can be visualized using silver stain (Fig. 3.14).

74 Fig. 3.14: Diagram Gel showing bands separated by SDS-PAGE.

Unit 3 Techniques in Molecular Biology
Two-dimensional gel electrophoresis
In this type of electrophoresis, the mixture of proteins can be separated using
two different properties of the molecules. Proteins are first separated in a
tubular gel according to their isoelectric point via isoelectric focusing
technique. The gel is removed and placed on the top of a slab of SDS
saturated polyacrylamide and subjected to SDS- PAGE. The proteins move
into the slab gel and get separated on the basis of molecular mass. The
proteins are removed from the gel and digested into peptide fragments that
can be analyzed by mass spectrometry.

This type of gel electrophoresis is used to detect changes in the proteins

present in a cell under different conditions. The technique is suitable for
distinguishing proteins that have high molecular mass, are highly hydrophobic
or present in very low copy numbers per cell.

Applications
Electrophoresis technique is used for the analysis of biological or other
polymeric samples. The electrophoresis techniques are used to separate
various types of biomolecules, analyze their characteristics and study their
interaction with a molecule of interest. Biomolecules such as DNA, RNA and
proteins are separated using this technique.

i) The gel electrophoresis technique helps to visualize, identify and

distinguish molecules such as nucleic acids.

ii) Small RNA or DNA molecules of few hundred nucleotides are separated
by PAGE.

iii) SDS-PAGE is a technique commonly used for identification and

separation of proteins.

iv) Pulse field electrophoresis is used for separation of DNA molecules

greater than 25kb.

v) Agarose Gel Electrophoresis is most commonly used for separation and

identification of DNA molecules.

vi) Denaturing PAGE (polyacrylamide gel electrophoresis) is commonly

used for separation of RNA.

SAQ 3
a) Answer in one word :

i) An instrument that measures the amount of light absorbed by a

sample when a beam of light is passed through it.

ii) In spectrophotometer the concentration of coloured and colourless

compounds can be determined by measuring this.

iii) In this type of centrifugation the components of the sample get

distributed into different layers according to their density i.e.
separation occurs under the influence of gravitational force.

iv) This type of centrifugation results in separation of cellular

organelles. 75
Block 1 Techniques in Cell Biology
v) A high concentration of sucrose or cesium chloride is used for the
separation of molecules.

vi) In this technique molecules such as proteins, amino acids,

nucleotides are separated on the basis of differences in their net
charge when placed in an electric field.

vii) The solution obtained after centrifugation.

b) Fill in the blanks.

i) ……………. solution is used in the density gradient centrifugation.

ii) A high concentration of ……………… is used in ultracentrifugation.

iii) During electrophoresis the gel can be stained using dye

…………………….. .

iv) In two dimensional gel electrophoresis, proteins are first separated

according to their ……………………. .

v) Gel electrophoresis is also used to separate nucleic acids of

different ………………….. .

c) State whether the statements are ‘True’ or ‘False’.

i) In spectrophotometry, the amount of light absorbed by a colored

solution is directly proportional to the concentration of the solution.

ii) In ultracentrifugation, the components get separated on the basis

of their speed.

iii) An incident beam of monochromatic X-rays interacts with a target

material in X-ray diffraction analysis.

iv) The movement of proteins in a polyacrylamide gel electrophoresis

depends on the size of the molecules.

v) SDS binds to the hydrophobic regions of the protein molecules.

vi) Gel electrophoresis is also used to separate nucleic acids of

different charges.

3.6 SUMMARY
• A spectrophotometer is an instrument used to measure absorbance of
the compound at different wavelengths. It is commonly used for the
determination of the concentration of coloured and colourless
compounds by measuring their optical density or absorbance.

• In centrifugation technique the contents of the sample get separated into

various layers according to their density. The technique is used to isolate
cell organelles. It is based on the principle that particles of different size
and shape travel toward bottom of the centrifuge tube with respect to the
76 surrounding medium when placed in a centrifugal field.
Unit 3 Techniques in Molecular Biology
• X-Ray Diffraction is a method used to analyze the structure of crystalline
materials. The protein crystals are bombarded with a fine beam of X-
rays. The radiation gets scattered (diffracted) when the electrons strike
the proteins atoms. The diffraction produced determines the structure
within the protein and other crystalline materials.

• Electrophoresis is a technique by which the molecules such as proteins,

amino acids, nucleotides are separated on the basis of differences in
their net charge when placed in an electric field. The separation of the
molecules depends on the ability of the charged molecules to migrate
when placed in an electric field.

• SDS-PAGE is the electrophoresis technique that facilitates better

separation and analysis of proteins. The gel is prepared by
polymerization of monomers. The pore size in the gel regulates the
passage of proteins. The proteins are held in by the solution having
negatively charged detergent called sodium dodecyl sulphate (SDS).
SDS binds to the hydrophobic regions of the protein molecules and
causes them to unfold into extended polypeptide chains. When the SDS
treated proteins are passed through the gel, proteins migrate towards
the positive terminal electrode.

3.7 TERMINAL QUESTIONS

1. Describe the applications of:

a) Spectrophotometer

b) X-ray diffraction analysis

c) Ultracentrifugation

2. Enlist the differences between density gradient and differential

centrifugation.

3. What governs the movement of proteins in Polyacrylamide gel

electrophoresis (PAGE)?

4. Write short notes on:

a) Two dimensional gel electrophoresis

b) Principle of centrifugation

3.8 ANSWERS
Self-Assessment Questions
1. a) The working principle of the spectrophotometer is based on Beer-
Lambert’s law. There is a linear relationship between absorbance
and concentration of an absorber of electromagnetic radiation. It
states that the amount of light absorbed by a coloured solution is
directly proportional to the concentration of the solution and the
length of a light path through the solution.

b) Refer to Fig. 3.2. 77

Block 1 Techniques in Cell Biology
2. a) Beer-Lambert’s law

b) optical density or absorbance

c) Relative centrifugal force (RCF)

d) centrifugal force

e) microfuge

3. a) i) spectrophotometer

ii) optical density or absorbance

iii) density gradient centrifugation

iv) differential centrifugation

v) ultracentrifugation

vi) electrophoresis

vii) homogenate

b) i) sucrose

ii) sucrose or cesium chloride

iii) Coomassie brilliant blue

iv) isoelectric point

v) molecular mass

c) i) True; ii) False; iii) True; iv) False; v) True; vi) False

Terminal Questions
1. a) The spectrophotometer is used for determining the concentration
of compounds by measuring their optical density or absorbance.
The spectrophotometer works on the principle of Beer-Lambert’s
law. This law states that the amount of light absorbed by a
coloured solution is directly proportional to the concentration of the
solution and the length of a light path through the solution.

b) The protein crystals are bombarded with a fine beam of X rays.

The radiation (X-rays) get scattered (diffracted) when the electrons
of the protein atoms strike an electron sensitive detector placed
behind the crystal. An incident beam of monochromatic X-rays
interacts with a target material.

c) The molecules in this case can be separated on the basis of their

buoyant density. The band of highest buoyant density is at the
bottom and lowest at the top.

2. The main difference between differential and density gradient

centrifugation is that fractionation is carried out based on the size in
differential centrifugation whereas fractionation is carried out based on
78 the density in density gradient centrifugation.
Unit 3 Techniques in Molecular Biology
3. It is a technique by which the molecules such as proteins, amino acids,
nucleotides are separated based on differences in their net charge when
placed in an electric field. The separation of the molecules depends on
the ability of the charged molecules to migrate when placed in an electric
field. The technique is used in purification of enzymes.

4. a) Refer to Section 3.5.

b) Refer to Section 3.3.

Acknowledgement for figures

Fig. 3.1 : https://www.google.co.in/imgres?imgurl=https%3A%2F%2Fptop.only.wip.la%3A443%2Fhttps%2Fww
w.unicosci.com%2Fmedia%2Fcatalog%2Fproduct%2Fcache%2
Fimage

Fig. 3.3 : https://www.google.co.in/imgres?imgurl=https%3A%2F%2Fptop.only.wip.la%3A443%2Fhttps%2Fus.v

wr.com%2Fstibo%2Fbigweb%2Fstd.lang.all%2F77%2F39%2F4
677739.jpg

https://www.google.co.in/imgres?imgurl=https%3A%2F%2Fptop.only.wip.la%3A443%2Fhttps%2Fimg.
medicalexpo.com%2Fimages_me%2Fphoto-g%2F131159-
16310271.jpg

Fig. 3.6 : https://www.google.co.in/imgres?imgurl=https%3A%2F%2Fptop.only.wip.la%3A443%2Fhttps%2Fww

w.himac-science.com %2Fproducts% 2Fhimac% 2Fultra%
2Fcpnx%2Fcpnx.jpg

Fig. 3.13 : https://www.google.co.in/imgres?imgurl=https%3A%2F%2Fptop.only.wip.la%3A443%2Fhttps%2Fww

w.syngene.com%2Fwp-content%2Fuploads%2Fingenius-gel-
doc-system-1-1.jpg

Fig. 3.14 : https://www.google.co.in/imgres?imgurl=https%3A%2F%2Fptop.only.wip.la%3A443%2Fhttps%2Fww

w.researchgate.net%2Fprofile%2FJuwartina-Ida-
Royani%2Fpublication%2F328710018%2Ffigure%2Ffig1%2FA
S%3A688711058591744%401541212920226%2FSDS-PAGE-
sodium

You tube videos

https://www.youtube.com/watch?v=xgxFBQZYXlE

https://www.youtube.com/watch?v=MhJT9yjnl88

https://www.youtube.com/watch?v=bYwq5oNZmq4

https://www.youtube.com/watch?v=4OJAzQsZnbo

79