ISLAMIC INTERNATION MEDIACL COLLEGE

DEPARTMENT OF BIOCHEMISTRY

SGD TOPIC: METHODS TO STUDY CELL BIOCHEMISTRY

LEARNING OBJECTIVES:

Describe the following scientific methods to study the cell biochemistry:

1. Tissue homogenization (Principle, Procedure)

2. Centrifugation (Principle, parts of centrifuge machine ,procedure)
3. Electrophoresis ((Principle, parts of electrophoresis tank, procedure)

TISSUE HOMOGENIZATION
 INTRODUCTION

To purify or characterize an intracellular protein, it is important to choose an efficient method for

disrupting the cell or tissue that rapidly releases the protein from its intracellular compartment into
a buffer that is not harmful to the biological activity of the protein of interest.

One of the most widely used methods for disrupting soft tissues is homogenization. Homogenization
to break tissues down into their constituent pieces is a common first step in the lab. Depending on
the desired constituent parts, different techniques may be used. For more thorough
homogenization, blending of the tissue is often done first and a disruptor is then used to break the
tissue down even further.

Homogenizers can have dedicated functions, such as a tissue-specific homogenizer, or more general
functions. It is a technique of isolating cellular components. Cellular components are obtained by
differential centrifugation of cells, after they have been gently broken, i.e. homogenized by any of
the method.

 DEFINITION OF HOMOGENIZATION
Homogenization is a process whereby a biological process is brought to a state such that all fractions
of the sample are equal in composition.

 PRINICIPLE OF TISSUE HOMOGENIZATION

A homogenizer is a laboratory equipment which is used for the homogenization of various materials.
Cell fractionation is done by homogenizer to release organelles from cell.
 WHAT IS THE PURPOSE OF HOMOGENIZATION?

Homogenization is the process by which a sample is broken into identical parts so that removing one
portion of it does not disrupt and still accurately reflects the remaining sample’s molecular
composition. In many instances, it is also used to thoroughly mix together naturally immiscible
substances; when a sample is reduced to tiny, uniform pieces:

1. To Reduce Particle Size

First and foremost, the purpose of homogenization is to reduce the size of the particles making up a
sample. During homogenization, many samples can be reduced to nanometer-sized particles. This
makes it easier for manufacturers to produce things like adhesives, resins, paints and creams/lotions
and guarantee that they work.

2. To lyse cells

For the molecular and cellular biology fields, one of the most common purposes of homogenization
is to breach the cell wall and/or membrane and expose the intracellular contents of cells. This is
necessary when scientists need to make biological samples for further study, experimentation and
research.

3. To Kill Pathogens

One the added benefits of many homogenization techniques is the destruction of pathogens within

a sample. There are several kinds of homogenizers (mechanical, high pressure and ultrasonic), each
using one or more specific types of force

4. To Facilitate Stable Emulsions and Dispersions

The purpose of most homogenizers is to mix substances well. Reducing the size of the particles in a
substance allows it to more readily and easily combine with the particles of another substance. It
also allows all of the particles to remain mixed for a longer period of time. This is especially
important for products that need to be consistent in appearance, taste, texture, viscosity and/or
nutritional value for the length of their shelf lives, like cosmetics, food and beverage items and
pharmaceuticals.

 PROCEDURE

Tissue homogenization in solution is often performed simultaneously in with cell lysis. To prevent
lysis however, the tissue (or collection of cell e.g. from cell culture) can be kept at temperatures
slightly above zero to prevent autolysis, and in an isotonic solution to prevent osmotic damage.

 SAMPLE PRESERVATION, CONTAINERS, HANDLING, AND STORAGE

 Sample Storage and Handling
 Tissue samples (or specimens) must be protected from light and kept frozen below -100C from the
time of collection until the homogenization process.

Tissue samples and sample homogenates in glass jars must be stored in a freezer free of all potential
contaminants.

Before and after tissue sample homogenization, tissue samples must be protected from light and
kept frozen below -10oC for the periods

Tissue samples, homogenates, and sample extracts must be stored separately from standards.

Holding Times- Homogenization of tissue samples received at 4 o ± 2 o C or above shall be completed within
seven (7) days of sampling. Tissue samples received at -10 oC do not have any holding time requirements
until thawed for processing.

 EQUIPMENT/APPARATUS

The following equipment/apparatus are required:

 Variable speed laboratory blenders Blending

 containers - stainless steel (SS) with SS lids of various sizes [40, 100, 250, and 500 milliliters (mL)]
depending on sample size
 grinder
 Stainless steel and/or Teflon coated forceps,
 spatulas,
 spoons, and scoops
 Stainless steel knife,
 cleaver, and
 scissors
 Teflon cutting board
 Stainless steel trays
 Freezer, capable of maintaining a temperature of less than (-10C)
 Balance, capable of weighing 200 grams (g) to the nearest 0.01 g
 Glass collection jars with Teflon-lined lids (variable sizes)
 Coolers Test tube brushes TurboVap II concentrator, with rack and concentrator cells
 REAGENTS

 DIFFERENT METHODS FOR PERFORMING TISSUE HOMOGENIZATION

Four different tissue homogenization techniques for their ideal cell types, batch sizes, and potential
benefits/caveats are discussed below:
1. Chemical Homogenization
 Most disruption methods use some form of lysis buffer or chemical to provide stability when isolating
specific biomolecules. Yet some chemicals can be used alone to effectively homogenize tissues. For
example,

a. Surfactants and detergents target biological membranes by disrupting the hydrophobic/hydrophilic

interface, and are well-matched with various bacterial species.
b. Enzymes also tackle the cell membrane and/or wall, and can be effectively used as a first step in obtaining
tissue extract.
Chemical homogenization is preferable for small samples, as the materials cost can become overwhelming
for industrial-sized products

2. Freeze-Thawing

Frequently employed to disrupt bacterial and mammalian cells, freeze-thawing is nearly as simple as it
sounds. A tissue suspension is first frozen and then thawed at room temperature. Ice crystals that formed
during the freezing process contract as the sample thaws, which ruptures the cell’s membrane. Although it
effectively releases recombinant cytoplasmic proteins, the freeze-thaw process requires multiple cycles and
requires significant amounts of time.

3. Mechanical Homogenization

Encompassing equipment like rotor stators and high pressure homogenizers, mechanical homogenization
works by using pressure and/or force(s), instead of heat, to mechanically disrupt cells. Scientists often turn
to this technique because of its ability to be easily scaled, as well as its quick process and
uniform/consistent results.

4. Ultrasonic (high frequency sound waves) Homogenization

Ultrasonic homogenizers, also known as sonicators, rupture tissues through a combination of cavitation and
ultrasonic waves. This technique is ideally matched for suspended cellular/subcellular structures, as well as
for shearing DNA. However, because it generates a significant amount of heat, ultrasonic homogenization is
only appropriate for tissues and molecules that will not be affected by temperature increase. 

Applications

 Tissue Homogenization.
 Cell Disruption.
 Tissue Dissociation / Cell Isolation.
 Cellular Organelle Extraction.
 Microorganism Extraction.
  Particle Size Reduction.
 Nanoparticle Creation.
 Creating Emulsions.

able 2.1.  Summary of different homogenization methods and corresponding techniques.

Method type Technique

Mortar and pestle, Bessman pulverizer

Grinding/shearing
CryoPrep freeze fractionation

Blending Rotor–stator, blender

Sonication Bath or probe sonicator, focused acoustics ultrasonicator

Bead beating Bead mill, multidirectional bead beater

Enzymatic digestion Enzymatic (protease, collagenase)

CENTRIFUGATION
 INTRODUCTION

Centrifugation is a technique used for the separation of particles from a solution according to their
size, shape, density, viscosity of the medium and rotor speed. A centrifuge is a device for separating
particles from a solution according to their density.

 PRINCIPLE OF CENTRIFUGATION:

A centrifuge is used to separate particles suspended in a liquid according to particle size and density,
viscosity of the medium, and rotor speed.

Within a solution, gravitational force will cause particles of higher density than the solvent to sink,
and those less dense than the solvent to float to the top. Centrifugation takes advantage of even
minute differences in density to separate particles within a solution.

As the rotor spins around a central axis, it generates a centrifugal force acting to move particles
away from the axis of rotation. If the centrifugal force exceeds the buoyant forces of liquid media
and the frictional force created by the particle, the particles will sediment.

Fig I: Centrifuge Machine Fig II: Procedure of centrifugation

Which factors have an influence on centrifugation?

 Density of both samples and solution
 Temperature/viscosity
 Distance of particles displacement
 Rotation speed

 CENTRIFUGE
 A centrifuge is a device that separates particles from a solution through use of a rotor. In biology,
the particles are usually cells, subcellular organelles, or large molecules, all of which are referred to
here as particles.
 There are two types of centrifuge procedures; one is preparative, the purpose of which is to isolate
specific particles, and the other is analytical, which involves measuring physical properties of the
sedimenting particles.
 As a rotor spins in a centrifuge, a centrifugal force is applied to each particle in the sample; the
particle will then sediment at the rate that is proportional to the centrifugal force applied to it. The
viscosity of the sample solution and the physical properties of the particles also affect the
sedimentation rate of each particle.
 At a fixed centrifugal force and liquid viscosity, the sedimentation rate of a particle is proportional
to its size (molecular weight) and to the difference between the particle density and the density of
the solution.

CENTRIFUGATION THEORY

In a solution, particles whose density is higher than that of the solvent sink (sediment), and
particles that are lighter than it float to the top.The greater the difference in density, the faster
they move. If there is no difference in density (isopyknic conditions), the particles stay steady

To take advantage of even tiny differences in density to separate various particles in a solution,
gravity can be replaced with the much more powerful “centrifugal force” provided by a centrifuge.
 TYPES AND USES OF CENTRIFUGES
The types of centrifuges are all based on the same technique but differ in their applications. The
main differences between them are the speed of rotation and the rotor design. The rotor is the
rotating unit in the device. Fixed-angle rotors hold samples at a constant angle, swinging head rotors
have a hinge that allows sample vessels to swing outward as the rate of spin increases, and
continuous tubular centrifuges have a single chamber rather than individual sample chambers.

 PARTS OF A CENTRIFUGE MACHINE: Centrifuges have three basic components:

  A rotor 
 A drive shaft 
 A motor

The rotor holds the tubes, bottles, or bags containing the liquids to be centrifuged. Different rotor
types and sizes, interchangeable with one another, can be mounted on the drive shaft, which
connects to the motor. The motor provides the power to turn the rotor. Usually, a cabinet
surrounds and supports these parts, and also protects the operator should a tube break or any
metal parts fail while the centrifuge is running. The operating controls and indicator dials for speed
and time are mounted on the cabinet. Most centrifuges have a brake system to bring the rotor to a
standstill shortly after the run is finished. Unlike the mechanical brakes on a car, the braking action
is electrical, the current to the motor is simply reversed. Many centrifuges are also refrigerated to
prevent delicate biological samples from getting warm.

 HOW TO BALANCE A CENTRIFUGE 

1. Ensure all sample tubes are evenly filled. If additional tubes are required for balancing, fill them with
water or a liquid of similar density to the sample, and ensure the mass is balanced to the nearest 0.1
grams.
2. For each tube inserted in the rotor, add a tube of equal weight directly opposite it. This will ensure
the center of gravity remains in the center of the rotor.
3. Rotate the rotor 90° and add two additional tubes directly opposite one another.
4. Repeat.

 APPLICATIONS/USES OF CENTRIFUGATION:

 Centrifuges are used in various laboratories to separate fluids, gases, or liquids based on density.
 In research and clinical laboratories, centrifuges are often used for cell, organelle, virus, protein, and
nucleic acid purification.
 An example of centrifuge use in a clinical setting is for the separation of whole blood components.
Different assays necessitate serum or plasma, which may be obtained with centrifugation.
 o Serum is obtained by letting a whole blood sample clot at room temperature. The sample is
then centrifuged and the clot is removed, leaving a serum supernatant.
o Unlike serum, plasma is obtained from whole blood that is not left to clot, and contains serum
along with clotting factors. To obtain plasma, a whole blood sample is collected in tubes
treated with anticoagulants. Following centrifugation, cells are removed and plasma
supernatant remains.

 To isolate macromolecules like DNA, RNA, proteins, lipids

ELECTROPHORESIS
 INTRODUCTION

Electrophoresis is based on the phenomenon that most biomolecules exist as electrically-charged

particles, possessing ionizable functional groups. Biomolecules in a solution at a given pH will exist
as either positively or negatively charged ions.

Electrophoresis is an electrokinetic process which separates charged particles in a fluid using a field
of electrical charge. Electrophoretic separation occurs when an electric field is applied between two
electrodes, cathode, and anode, which are submerged in a buffer solution.

It is most often used in life sciences to separate protein molecules or DNA and can be achieved
through several different procedures depending on the type and size of the molecules.

 PRINCIPLE OF ELECTROPHORESIS

When subjected to an electric field, ionized biomolecules will migrate at a different pace, depending
on the mass and the net charge of each particle in the solution—negatively-charged particle, anions,
 will migrate towards a positively charged electrode, or cathode, and cations, or positively-charged
particles, will be pulled towards a negatively-charged electrode called anode.

 TYPES OF ELECTROPHORESIS

The following are selected electrophoresis methods, based on different formats of electrophoresis.

1. Gel Electrophoresis
Gel electrophoresis is a laboratory method used to separate mixtures of DNA, RNA, or proteins
according to molecular size. In gel electrophoresis, the molecules to be separated are pushed by an
electrical field through a gel that contains small pores. The molecules travel through the pores in
the gel at a speed that is inversely related to their lengths. This means that a small DNA molecule
will travel a greater distance through the gel than will a larger DNA molecule.

The gel is cast into strips or slabs with slots or sample wells. Once it is completely polymerized, the
gel is submerged in an electrophoresis solution buffer, and samples are loaded into each well
before the electric current is applied to initiate electrophoretic separation (Walker, 2010). At the
end of the gel electrophoresis, components in the samples will be separated based on their mass 

Gel types
a. Polyacrylamide -  Generally, a low percentage of acrylamide gel (3%-15%) is used to separate
DNA and proteins. Higher percentage acrylamide gel (10%-20%) is commonly used in sodium
dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE), in which proteins are
separated in denatured condition, according to their size 

b. Agarose- A natural linear polysaccharide made of galactose and 3, 6-anhydrogalactose

chains, extracted from agar isolated from red seaweeds . Agarose, like agar, is stored as a
dry powder. To cast agarose gel, the agarose powder is dissolved in a relevant solution
buffer, heated, and allowed to cool to room temperature. Similar to polyacrylamide gel, the
concentration of the agarose in the solution buffer determines the pore size of the gel.
Agarose gel is commonly used at 0.8% (w/v) to 5% (w/v) to separate DNA and RNA
molecules. Agarose can be used in combination with SDS to separate high-molecular-weight
proteins, which can be problematic when separated using SDS-PAGE.

2. Pulsed-field Gel Electrophoresis (PFGE)

Pulsed-field Gel Electrophoresis (PFGE) is a variation of gel electrophoresis, in which two electrical
fields are periodically applied, in rotation, to the gel electrophoresis at different angles. This type of
electrophoresis is specifically designed for the separation of chromosomes, which are high-
molecular-weight DNA molecules of over 20 kilobases.
 3. Capillary Electrophoresis (CE)

Capillary electrophoresis, also known as High-Performance Capillary Electrophoresis (HPCE), is a

type of electrophoresis performed in a narrow capillary immersed in an electrolyte buffer. It is the
only type of electrophoresis capable of performing all four types of electrophoreses (Heiger, 2000).
The capillary is typically 20-30 centimeters long and possesses a 25-75 micrometer inner diameter.

Electrophoretic separation is initiated when a sample is injected into the capillary, either by high
voltage or by pressure, and high electric fields are applied across the capillary. Components in the
sample are separated along the length of the capillary, based on the format of electrophoresis
performed.

4. Immunoelectrophoresis

Immunoelectrophoresis is a type of electrophoresis that separate antigens, including proteins and

peptides, based on their reaction and specificity to antibodies, or immunoglobulins (Ig). 

5. Affinity Electrophoresis

Affinity Electrophoresis is a type of electrophoresis that separates a biomolecule that interacts with
or binds to another molecule for which it has an affinity. It makes use of the phenomenon that the
electrical mobility (µ) changes when a biomolecule, including nucleic acids, proteins, peptides, and
polysaccharides, binds to another molecule, and this change in the electrical mobility will be
reflected in the electrophoretic pattern.
 INSTRUMENTATION

All types of electrophoresis separate charged particles while they are submerged in a solution
buffer. All forms of electrophoresis require a

  power supply
  electrophoresis unit, commonly referred to as an electrophoresis chamber.
 The power supply provides the electric current to the chamber that propels the
electrophoretic separation.
 The chamber is composed of two opposite electrodes, cathode and anode
 and of a buffer solution reservoir, in which the samples and the separation thereof take
place
 PROCEDURE
 The +ve and –ve leads are connected to a chamber and power supply where the voltage is set.
 Turning on the power supply sets up the electric field and the – vely charged DNA samples will start
to migrate through the gel and away from the –ve electrode towards the electrode.
1. Stopping Electrophoresis and visualizing the DNA:
 Once the blue dye in the DNA samples has migrated through the gel far enough, the power supply is
turned off and the gel is removed and placed into an ethidium bromide solution.
 Ethidium bromide intercalates between DNA and is visible in UV light.
 The Ethidium bromide stained gel is then exposed to UV light and a picture is taken,
 DNA bands are visualized in from each lane corresponding to a chamber well.
 The DNA ladder that was loaded is also visualized and the length of the DNA bands can be estimated.
2. DETECTION METHODS

There are several approaches to detect and visualize the result of electrophoresis, each with a
different degree of sensitivity and complexity. Common detection methods are as follows 

 Staining, which uses a variety of dyes that can bind to the molecules of interest. For
example, Coomassie Brilliant Blue or silver staining can bind to proteins. Ethidium bromide This
approach is a common detection method for gel electrophoresis.

 Autoradiography, which can be used for detection if the molecules of interest are labeled with
radioisotopes. Compared to staining, autoradiography is more sensitive.
 Online detection, , is a part of automated instrumentation. Several detection mechanisms can be
employed for online detection such as detection of UV absorption, fluorescence, electric field, and
conductivity. 
APPLICATIONS/USES OF ELECTROPHORESIS:
1. Identification of abnormal patterns of plasma proteins in various disease processes
2. Identification/quantification of normal and abnormal protein bands
3. Identification and quantification of normal and abnormal hemoglobins
4. Quantification of lipoproteins
5. Identification of isozymes

FURTHER READING (BOOKS/LINKS)

1. Textbook of Medical Biochemistry by Chatterjea
2. Tissue homogenization: https://www.youtube.com/watch?v=nRXqKnmCWOo&feature=youtu.be
3. Centrifuge: https://www.youtube.com/watch?reload=9&v=NqVaMiTI8Uw
4. Electrophoresis: https://youtu.be/QEG8dz7cbnY