3. • Differential centrifugation (also known
as differential velocity centrifugation) is a
common procedure in biochemistry and cell
biology, which is used to separate organelles
and other sub-cellular particles based on their
sedimentation rate.
4. Principle
• In a viscous fluid, the rate of sedimentation of
a given suspended particle (as long as the
particle is more dense than the fluid) is largely
a function of the following factors:
• Gravitational force
• Difference in density
• Fluid viscosity
• Particle size and shape
5. Procedure
• Differential centrifugation can be used with intact particles
(e.g. biological cells, microparticles, nanoparticles), or used
to separate the component parts of a given particle.
• Using the example of a separation of eukaryotic organelles
from intact cells, the cell must first be lysed
and homogenized (ideally by a gentle technique, such as
dounce homogenization; harsher techniques or over
homogenization will lead to a lower proportion of intact
organelles).
• Once the crude organelle extract is obtained, it may be
subjected to a varying centrifugation speeds to separate
the organelles:
6. Typical differential centrifugation parameters for a biological sample
Sample input G force Time
Instrument
needed
Pellet contents
Supernatant
contents
Unlysed (eukaryotic) cells 100 x g 5 min
Benchtop fixed-
angle
centrifuge, or
swinging bucket
centrifuge
Intact
(eukaryotic)
cells,
macroscopic
debris
Varies
depending on
sample
Gently lysed cells (e.g. dounce homogenizer) 600 x g 10 min
Benchtop fixed-
angle
centrifuge, or
swinging bucket
centrifuge
Nuclei
Cytosol, non-
nuclei
organelles
Supernatant of previous row 15,000 x g 20 min
Benchtop fixed-
angle centrifuge
Mitochondria,
chloroplasts,
lysosomes,
peroxisomes
Cytosol, microso
mes (known as
post
mitochondrial
supernatant)
Supernatant of previous row
50,000 x g -
100,000 x g
60 min
High speed
fixed-angle
centrifuge, or
vacuum
ultracentrifuge
Plasma
membrane,
microsomal
fraction, large
polyribosomes
Cytosol,
ribosomal
subunits, small
polyribosomes,
enzyme
complexes
Supernatant of previous row
50,000 x g -
100,000 x g
120 min
Vacuum
ultracentrifug
e
Ribosomal
subunits,
small poly
ribosomes,
some soluble
enzyme
complexes
Cytosol
7. • It is the most common type of centrifugation employed.
• Tissue such as the liver is homogenized at 32 degrees in a
sucrose solution that contains buffer.
• The homogenate is then placed in a centrifuge and spun at
constant centrifugal force at a constant temperature.
• After some time a sediment forms at the bottom of a
centrifuge called pellet and an overlying solution called
supernatant.
• The overlying solution is then placed in another centrifuge
tube which is then rotated at higher speeds in progressing
steps.
8. Applications
• To separate two miscible substances
• To analyze the hydrodynamic properties of macromolecules
• Purification of mammalian cells
• Fractionation of subcellular organelles (including
membranes/membrane fractions) Fractionation of membrane
vesicles
• Separating chalk powder from water
• Removing fat from milk to produce skimmed milk
• Separating particles from an air-flow using cyclonic separation
• The clarification and stabilization of wine
• Separation of urine components and blood components in forensic
and research laboratories
• Aids in the separation of proteins using purification techniques such
as salting out, e.g. ammonium sulfate precipitation.
