Cellular fractionation &
Biochemical Markers
Dr Sabeen Khalid
Cell fractionation

Cell fractionation is a method to separate
subcellular components, and isolate
organelles and other subcellular
components from one another
Steps of fractionation

Extraction

Homogenization

Centrifugation
Extraction
It is the first step toward isolating any
sub-cellular structures
 In order to maintain the biological activity
of organelles and bio-molecules, they
must be extracted in mild conditions
(employment of aqueous solution,
avoidance of extreme of pH, osmotic
pressure, high temperature)

A common solution for extraction of
organelles consist of
1.
2.
3.
Sucrose, 0.25 mol/l (isoosmotic),
TRIS HCl buffer to adjust pH at 7.4
K+ & Mg++ ions at near physiologic
concentrations
Organic solvents are also used for
extraction of lipids & nucleic acids
Homogenization
The suspended cells are then
disrupted by the process of
homogenization
 Its purpose is to break cell
membranes open and release
organelles into the isotonic buffered
solution
 When the inner contents of a cell leak
out into the environment, it is called lysis





Homogenization can be accomplished by
mechanical or chemical means
In mechanical breakage, the cell solution
is placed in a high-speed blender and then
filtered
In chemical homogenization, enzymes or
detergents are added to the solution to
dissolve the lipid bilayer that surrounds and
protects the cell
Once the cell solution has been
homogenized, and the organelles leak out
due to lysis, the solution becomes a
homogenate
Centrifugation
In the classic method, a series of 03
different centrifugation steps at
successively greater speed yield a pellet &
supernatant
 The supernatant from each step is
subjected to centrifugation in the next
step
 This procedure provides three pellets,
named the nuclear fraction,
mitochondrial fraction, &
microsomal fraction

o Nuclear
fraction → contain nuclei &
unruptured cells
o Mitochondrial
fraction → contain
mitochondria, lysosomes & peroxisomes
o Microsomal fraction → contain mixture
of free ribosomes, smooth ER, rough ER
Marker enzymes/ biochemical
markers

A marker enzyme is one that is almost
exclusively confined to one particular
organelle

The marker thus can serve to indicate the
presence or absence of the organelle in
any particular fraction in which it is
contained
Organelle / fraction
Marker
Plasma membrane
• Na+-K + ATPase
• 5, - Nucleotidase
Golgi apparatus
Galactosyl transferase
Peroxisomes
• Catalase
• Uric acid oxidase
Cytosol
Lactate dehydrogenase
Mitochondria
Glutamic dehydrogenase
Endoplasmic reticulum
Glucose-6- phosphatase
Nucleus
DNA
Ribosomes
RNA
Lysosomes
Acid phosphatase