Biochemistry and Cell-biology owe a lot of their existence to the

Subcellular fractionation techniques.

Subcellular fractionation refers to disintegrating intact cells into their

integral parts, the cellular organelles.

For cell biologists, the key objective is to isolate each cellular organelle to a
high degree of purity even if the quantify is small.

This is necessary for undertaking electron microscopic studies to reveal

ultrastructure of organelles.

On the other hand, biochemists are more interested in macromolecular

composition (such as proteins, lipids and carbohydrates) and hallmark
enzymes of each subcellular fraction and therefore they seek pure fractions
in sufficient quantity.
Subcellular compartments/organelles vary in physical parameters such as
mass, size and density.

Differential centrifugation exploits the sedimentation behaviour of particles

varying in their densities. The particles are separated based on their differential
sedimentation velocities in a medium of considerably small density (ρ2) and low
viscosity.

The density of the medium is critical because a high-density medium will prevent
sedimentation of cellular organelles and the organelles will float as predicted by the
equation given below.
N =Sedimentation velocity
d=diameter of distance
ρ1=density of particle
ρ2=density of medium
g=centrifugal force
µ=Viscosity of medium

Often sucrose (0.25M) is used as a medium, this is because sucrose has the
‘right’ density to bring about sedimentation of cellular organelles. Moreover,
sucrose at 0.25 M doesn’t have a high osmolarity and viscosity. The consideration
of osmolarity is important because hyperosmotic concentrations of the medium can
lead to osmotic-shrinkage of organelles.
The differential centrifugation leads to separation of cellular lysate into a pellet and a
supernatant fraction. At each step, the pellet fractions are made up of heavier
fractions that settle and make a pellet at a given ‘g-force’ and time interval whereas
supernatant is made of parts that could not be pelleted at that g-force and time
values.

Differe
Repeate
higher s
of cells i
the vario
in the fig

low spee
minutes

medium
minutes

high spe

very high
hours

1/13/04 Liz Brandon-U

Equilibrium density centrifugation.

Equilibrium density centrifugation also known as gradient centrifugation is often

employed to separate cellular components that have closely related densities.

A non-ionic medium of low density is chosen which also has a low osmolarity and
viscosity for similar reasons explained earlier. Usually, Sucrose and Glycerol have
been medium of choice but lately synthetic mediums such as Nycodenz,
Iodixanol and Percol have been also used with great success and better resolving
ability.

Sucrose and glycerol are limited by the fact that if their concentrations are increased it
leads to a quick jump in osmolarity which is not suitable for obtaining intact organelles.

Even at high concentrations Percol, Nycodenz and Iodixanol don’t have a sharp
change in osmolarity.
For equilibrium density centrifugation, the
samples are spun at high ‘g-force’ values
(40,000-50,000) for almost 60 minutes, this
generate zones of varying densities inside
the tube and the organelles or subcellular
fractions are separated on basis of their
densities in the gradient medium, each
organelle in a zone equal to its density.
 COMPILED AND CIRCULATED BY BANGAMOTI HANSDA, ASSISTANT PROFESSOR,
DEPARTMENT OF BOTANY, NARAJOLE RAJ COLLEGE

COMMON MARKERS FOR ORGANELLES

BOATNY: SEM-VI, PAPER-DSE4T: ANALYTICAL TECHNIQUES IN PLANT SCIENCS, UNIT 2T: CELL FRACTIONATION