Cell fractionation is a process that separates cellular components while maintaining their functions, involving tissue disruption and centrifugation. Tissue disruption can be achieved through methods like homogenization, sonication, and osmotic lysis, while centrifugation uses relative centrifugal force to separate components based on size and density. Differential centrifugation progressively isolates cellular components by sequentially spinning a lysate at increasing forces, producing pellets of mixed components and supernatants for further processing.

1. METHODS OF CELL
FRACTIONATION

2. • Cell fractionation is the process used to
separate cellular components while preserving
individual functions of each component.
• It is a process of producing pure fractions of
cell components.
• The process involves two basic steps namely:
disruption of the tissue and lysis of the cells,
followed by centrifugation.

4. Tissue Disruption and Lysis
• The first step in cell fractionation is tissue disruption and
cell lysis.
• The objective is to disaggregate the cells and break
them open with minimal or no damage to the cellular
fraction of interest.
• Tissues can be broken up and cells lysed in a number of
ways.
• The three basic methods of breaking up the tissues and
cells are:
– homogenization,
– sonication, and
– osmotic lysis.

5. • The particular method one chooses depends on the
tissue, the cell type, and the particular cell fraction of
interest.
• Most animal and plant tissues must be homogenized.
• Homogenization involves the use of a mechanical
homogenizer, like a blender or a mortar and pestle, to
break the tissue apart and lyse the cells.
• Sonication involves the use of ultrasound to disrupt the
cells. It is often used when prokarytic cells are to be lysed.
• Osmotic lysis is use when dealing with cells that are
vulnerable to osmotic stresses. Red blood cells are a
perfect example of a cell that can easily be lysed through
osmotic stress.

7. Lysing Mammalian Red Blood Cells
• Mammalian red blood cells are very sensitive to the
tonicity of the surrounding fluid.
• In vivo, mammalian red cells are bathed in isotonic
plasma, in which case there is no net osmosis and the cell
neither shrinks or swells.
• However, to lyse mammalian red blood cells as part of a
cell fractionation process, one can simply add cells to a
hypotonic fluid.
• The fluid must contain an adequate concentration of
physiological salts to buffer the solution at a physiological
pH so the plasma membrane remains functional.

8. Centrifugation
• The second step in the cell fractionation process
is centrifugation.
• Most of the cellular components in a cell lysate will
eventually, given time, settle to the bottom of a tube.
To accelerate this process, the lysate can be subjected
to centrifugation.
• In centrifugation, the lysate is rotated at a certain
speed (expressed as rotations per minute (RPM)).
• This rotation imposes a force on the particles
perpendicular to the axis of rotation.

9. • The force is called a relative centrifugal force (RCF),
expressed as a multiple of the force of Earth's
gravitational force (x g).
• For example, an RCF of 1000 x g is a force 1000 times
greater than Earth's gravitational force.
• When a particle is subjected to centrifugal force, it will
migrate away from the axis of rotation at a rate
dependent on the particle's size and density.

10.  That part of the centrifuge that holds the
centrifugation tubes is called the centrifuge
rotor.
 There are three type of centrifuge
rotors: fixed angle rotors, swinging bucket
rotors, and vertical rotors.
 Fixed-angle and swinging-bucket rotors are
the most commonly used. In a fixed-angle
rotor, the centrifuge tubes are spun at a
fixed angle, which is usually approximately
35 degrees.
 Fixed angle rotors are most commonly used
for pelleting cells and subcellular
components.
 With swinging-bucket rotors, the tubes are
free to swing-out perpendicular to the axis
of rotation as the rotor rotates.

12. • Differential centrifugation is one of two major types of
centrifugation schemes.
• Differential centrifugation is the sequential centrifugation
of a cell lysate at progressively increasing centrifugation
force, isolating cellular components of decreasing size and
density.
• The separation of the cellular components is based solely
on their sedimentation rate through the centrifugation
medium, which, in turn, is dependent on the size and
shape of the cellular components.
Differential Centrifugation.

13. HIGH
SPEED
CENTRIFUGATION
SUPERNATANT
2
 In differential centrifugation, each
centrifugation step results in the
production of a pellet, usually
containing a mixture of cellular
components of the same size and/or
density.
 The fluid resting above the pellet,
the supernatant, can be removed and
subjected to additional centrifugations to
generate pellets containing other cellular
components of lesser size and / or density.