Cell Fractionation

CE L L
FRA CT I O NATI O
N
Learning objectives:
Define cell fractionation
Explain the purpose of cell fractionation
Describe and explain the steps of cell fractionation

C E L L F R A C T I O N AT I O N
– a technique for separating cellular components while keeping

their distinct functions
– also called subcellular fractionation

Why is there a need to isolate the organelles when
there is electron microscopy to visualize the details of
the cell?
Electron microscopy provides information on the structure and function
of cellular organelles but fail to provide much insight into the molecular
composition of these structures.
Primary objective of cell fractionation:
To separate components and organelles, so that the structures,

functions, and molecular compositions of isolated components may
be studied.
S T E P S O F C E L L F R A C T I O N AT I O N
• Extraction
• Homogenization
• Filtration
• Centrifugation
1. EXTRACTION
• sample tissue are placed under mild conditions (known

as cell-free systems) before being used to protect it’s
integrity
• cold, isotonic, buffer solution

• Ice-cold (at 0-40°C)
– reduce the enzymatic activity that breaks down the organelle
• Isotonic solution (sucrose (0.25 mol/L) with a pH of 7.0)
– prevent water from moving into the organelles via osmosis
• Buffered (adequate pH and salt content)
– prevent organelle proteins from becoming denatured
2 . H O M O G E N I Z AT I O N
 breaking the cell membranes open and releasing organelles
 making sample to a state such that all fractions of the sample are
equal/uniform in composition
 pioneered by Albert Claude and Christian De Duve (1950s and
1960s)
 can be accomplished by mechanical or chemical methods,
sometimes scientists may incorporate both
 Mechanical Homogenization
– uses pressure and/or force(s) to mechanically disrupt cells

– grinding, blending, shearing, and shocking
– most common for its efficiency and ability to alter sample’s
physical structure while maintaining it’s biological composition
Homogenizer
–a laboratory device used to homogenize tissue/cell sample
–invented and patented by Auguste Gaulin in 1899 (described the
process of homogenizing milk)
–
Colloid mill Rotor-stator Bead mill Lab blender

 Chemical Homogenization
– enzymes or detergent added to the solution to dissolve the cell
membrane
– preferred for small sample
Other types of homogenization:
 Ultrasonic Homogenization (sonication)
– uses sound energy to disrupt tissues/cells
 Manual Homogenization
– mortar and pestle
– the organelles leak out due to lysis,
– various membranous organelles become fragmented and form spherical
membranous vesicles
– the solution becomes a homogenate
3 . F I LT R AT I O N
– homogenate is then filtered through a gauze

– a solution (known as the filtrate) that contains a mixture
of organelles
3 . C E N T R I F U G AT I O N
– applied centrifugal force field to separate the components according

to density and/or particle size
– a series of various sequential centrifugation at increasing centrifugal

forces
Centrifuge
As long as they are more dense than the surrounding medium,
particles of different size and shape travel toward the bottom of a
centrifuge tube at different rates when placed in a centrifugal field.
The smaller the subcellular component, the greater is the

centrifugal force required to sediment.
Types of Centrifugation Techniques:
 Density gradient centrifugation

 Differential centrifugation
 Density gradient Centrifugation
― separating based on size and mass density
― reagent is used to assist in isolation or separation of the

cells
― speed up the process, increase the purity
― able to sort particles of a much smaller size

 Differential Centrifugation
― most common and simplest form
― based on their size, density, and shape
― involves multiple centrifugation stages at increasing centrifugal

force
― the larger, denser particles have a higher rate of sedimentation

STEPS OF DIFFERENTIAL
CENTRIFUGATION
1. A centrifugation of homogenate at low speed.
– pellet is separated from the sedimented that is the nucleus
2. Supernatant is subjected to centrifugation at medium speed

– pellet is separated that is mitochondria
3. Supernatant at high speed

– pellet that has been sedimented contains lysosomes
4. Supernatant at very high speed

– pellet is separated from the sediment contains microsomes
The supernatant resulting from the final stage is the cell-free cytosol.
THANK YOU
!
References:
https://www.savemyexams.co.uk/a-level/biology/aqa/17/revision-notes/2-cell-structure/2-2-the-microscope-in-cell-studies/2-2-6-cell-fractionati
on--ultracentrifugation/
https://microbiologynote.com/differential-centrifugation/

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CE L L

Define cell fractionation

Explain the purpose of cell fractionation

Describe and explain the steps of cell fractionation

– a technique for separating cellular components while keeping

– also called subcellular fractionation

To separate components and organelles, so that the structures,

• sample tissue are placed under mild conditions (known

• cold, isotonic, buffer solution

– uses pressure and/or force(s) to mechanically disrupt cells

Colloid mill Rotor-stator Bead mill Lab blender

– homogenate is then filtered through a gauze

– applied centrifugal force field to separate the components according

– a series of various sequential centrifugation at increasing centrifugal

The smaller the subcellular component, the greater is the

 Density gradient centrifugation

― separating based on size and mass density

― reagent is used to assist in isolation or separation of the

― speed up the process, increase the purity

― able to sort particles of a much smaller size

― most common and simplest form

― based on their size, density, and shape

― involves multiple centrifugation stages at increasing centrifugal

― the larger, denser particles have a higher rate of sedimentation

2. Supernatant is subjected to centrifugation at medium speed

3. Supernatant at high speed

4. Supernatant at very high speed

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