Historical Introduction
In the early 1700s, Leeuwenhoek noted that avian
and amphibian blood cells contained a “clear area”
almost certainly corresponding to the structure we
now know as the nucleus. This represents probably the
earliest recognition that eukaryotic cells are not simply
sacs of protoplasm, but instead contain subcellular
structures. Although the concept of the nucleated cell
became Þrmly established in the 18th and 19th
centuries, the real beginning of subcellular
fractionation had to await the emergence in the mid1940s of the art form known as electronmicroscopy .
The electronmicroscope revealed much more
complexity than Leeuwenhoek could have
imagined. Concurrent with improvements in the
techniques of electron microscopy was the
development of methodologies for subcellular
fractionation. Through the 1950s, these parallel
approaches resulted in the discovery and isolation of
the major organelles that comprise the eukaryotic
cell. Finally, as biochemical functions were associated
with speciÞc subcellular compartments, a much
clearer picture of the eukaryotic cell began to
emerge, and with it the Þeld of modern cell biology.
What is cell fractionation
• Biologists need to study certain
organelles from a cell (the
mitochondria of a human cell or
the chloroplasts of a plant cell, for
example . Isolating these organelles
involves a variety of procedures
collectively called cell fractionation.
As a method for studying processes
within organelles, cell fractionation
has both advantages and
disadvantages.
• Cell fractionation is where a cell are broken up
and its components and organelles are
separated so that scientist can observe them
in isolated form .
• It can also be defined as : the separation of
homogeneous sets , usually organelles, from a
larger population of cells.
What we’ve learned so far using this
technique :
1.Mechanism of protein synthesis
2.DNA replication and transcription
3.RNA splicing
4.Muscle contraction
5.Microtubule assembly
6.Vesicular transport in the secretory pathway
7.Importance of mitachondria and chloroplasts
in energy interconversions .
Cell fractionation methods
Involve the homogenization or destruction
of cell boundaries by different mechanical or
chemical procedures, followed by the separation
of the subcellular fractions according to mass,
surface, and specific gravity
Steps of subcellular fractionation
1 . Homogenization
2 . Differential centrifugation
3 . Further separation and purification
by density gradient centrifugation
4 . Collection of fractions
5 . Analysis of fractions
1 . Homogenization
First the cells must be broken open. A variety of
different methods are available; the method
chosen depends on the type of experiment and the
type of sample (bacterial culture or mammalian
tissue sample, for example) Detergents like SDS or
Triton X disrupt the cell membrane so the contents
can flow out. Subjecting the cells to ultrasound
waves or sonicating them will also break them
open, as will agitation in the presence of metal or
glass beads. Blenders may work with tissue samples
but will not work with bacteria or other
microorganisms.
Homogenization or Cell Disruption
Chemical : alkali, organic solvents,
detergents
Enzymatic : lysozyme , chitinase
Physical : osmotic shock,
freeze/thaw
Mechanical : sonication ,
homogenization, French press
Chemical Disruption
• Detergents such as
Trition X-100 or NP40
can permeabilize cells
by solubilizing
membranes.
• Detergents can be
expensive, denature
proteins, and must be
removed after
disruption
Sonication
A sonicator can be
immersed directly
into a cell
suspension. The
sonicator is
vibrated and high
frequency sound
waves disrupt
cells.
Homogenization
• Cells are placed in a
closed vessel (usually
glass). A tight fitting
plunger is inserted and
rotated with a downward
force. Cells are disrupted
as they pass between the
plunger and vessel wall.
2 . Differential centrifugation
What is Centrifugation
Centrifugation is the process of isolating
components of a cell. There are two common
centrifugation techniques for separating
bacteria components.
Differential Centrifugation
Differential centrifugation is the process
where a homogenate (soup of tissue and
cells) undergoes repeat centrifugations and
increasing centrifugal force. Centrifugations is
the use of increased gravity to quicken the
precipitation of substances tot the bottom.
The tool used here is the centrifuge, "merrygo-round for test tubes" that spin at various
speeds.
The centrifuge separates the cell's parts
into pellet and supernatant. The pellet are the
large cell structures that are settled at the test
tube's bottom. The supernatant are smaller
parts of the cell suspending in liquid, the
supernatant is decanted and undergoes
another centrifugation. The process is
repeated and increases speed with each trial
to collect successively smaller parts of a cell in
pellets.
• These are both test tubes attached to a centrifuge. The first
picture is of a test tube that has undergone homogenization but is
about to undergo centrifugation . The second picture are the
results of the centrifugation and portrays the settling of large cell
parts.
The preparative ultracentrifuge
• The preparative ultracentrifuge. Sample is
contained in tubes that are inserted into a ring
of cylindrical holes in a metal rotor. Rapid
rotation of the rotor generates enormous
centrifugal forces, which cause particles in the
sample to sediment. The vacuum reduces
friction, preventing heating of the rotor and
allowing the refrigeration system to maintain
the sample at 4°C.
When a centrifugal force is applied to an
aqueous mixture, components of larger size
and density will sediment faster
Low speed centrifugation is used to separate
intact cells from medium
High speed centrifugation can be used to
separate subcellular components
Tow types of centrifuge are there
Fixed-Angle Centrifugation
Swinging-Arm Centrifugation
Method of Differential
Centrifugation:
• 1. Cut tissue in an icecold isotonic buffer.
It is cold to stop
enzyme reactions,
isotonic to stop
osmosis and a buffer
to stop pH changes.
• 2. Grind tissue in a
blender to break open
cells.
• 3. Filter to remove
insoluble tissue
• 4. Centrifuge
filtrate at low
speeds ( 1000 X g
for 10mins )
• This pellets the
nuclei as this is the
densest organelle
• 5. Centrifuge at
medium speeds ( 10
000 x g for 30
mins )
• This pellets
mitchondria which
are the second
densest organelle
• 6. Centrifuge at
high speeds ( 100
000 x g for 30
mins)
• This pellets ER,
golgi apparatus and
other membrane
fragments
• 7 Centrifuge at
very high speeds (
300 000 x g for
3hrs)
• This pellets
ribosomes
Buoyant density centrifugation
The buoyant density centrifugation involves viruses
with densities of 1.1-1.2 g/cm and a sucrose
gradient. The cell suspension is added to the top
of the sucrose gradient. In this centrifugation the
densest components move fastest down the tube
and stops at the sucrose density equal to its own.
The sucrose gradient bands at the bottom contain
cell components with high buoyant densities and
the components at the top have low buoyant
densities.
An illustration of the sucrose gradient and the
buoyant density centrifugation.
4 . Collection of fractions
Collecting Fractions-keeping samples pure
and intact
1.By hand: puncture sidewall of centrifuge tube
with needle and withdraw fractions through
syringe
2.Machine: gradient uploader; introduces very
dense, non-miscible medium into bottom of tube,
pushes fractions up to be collected from top
3.If no pellet, can collect fractions through hole in
bottom of tube
5 . Analysis of fractions
Analysis of fractions-need to identify and quantify the
purified fractions, so that they can be used successfully
in downstream applications
Methods:
1.Light or electron microscopy
2.Biochemical-determine presence of marker enzymes
3.Assay for a protein marker with an antibody (western)
4.Determine the protein concentration by using a
spectrophotometer, e.g. Bradford assay
5.Determine specific activity (the ratio of activity of
the enzyme of interest to the protein concentration
How is cell fractionation used in cell
biology?
• Scientists use this tool to increase their
knowledge of organelle functions. To be able to
do so they isolate organelles into pure groups,
such as isolating the mitochondria or the
nucleus.
For example, by centrifugation a specific cell
fraction was determined to have enzymes that
function in cellular respiration. This unknown cell
fraction was rich in mitochondria . Therefore
there researchers obtained evidence that helped
determine mitochondria were the site of cellular
respiration.
Investigating Cell Function
• Differential Centrifugation allows us to
look at each organelle within the cell
• We can look at the individual organelles
and study them in detail
• This helps to determine each organelles
function within the cell
Uses
• Separation of enzymes, hormones, RNA-DNA
hybrids, ribosomal subunits, subcellular
organelles, for the analysis of size distribution
of samples of polysomes and lipoprotein
fractions.
Represented by