Cell fractioning, principles and applications of centrifugation
The cell is a microscopic unit that contains various cell organelles like, nucleus, mitochondria, Golgi
complex, lysosomes etc. If one needs to study a particular organelle, or to examine their contents or to
isolate an enzyme to study its activities, the organelle needs to be isolated. To isolate the required
organelle in a purified state and in bulk it is done by subjecting the cells to differential centrifugation. It
is known that we can separate the heavier particles from lighter ones by centrifugation. When the cell
extracts are centrifuged the heaviest organelle will be the first to settle down, followed by the next heavier
one and finally the lightest organelle or substances will settle down.
The separation of the cell organelles can be done as they vary in mass, surface and specific gravity and
settle down at different speeds when subjected to centrifugation. Depending on the speed the standard or
ultracentrifuges are used.
Cell fractioning is the process by means of which the different cell organelles are separated by
differential centrifugation. The various cell organelles vary in density, mass, surface and specific
gravity. When subjected to centrifugal force they tend to settle down at different speeds. They can then be
separated easily and later purified. The speed at which each organelle settles down at different and can be
expressed in terms of Svedberg units (S). The ribosome units are labeled as 80S or 70S.
The principle
Cell purification is achieved by centrifugation. Centrifugation is done by an apparatus called a
centrifuge. It has balanced tubes attached to the opposite ends of arms rotating rapidly about a central
point. The cell extract is poured into the test tubes and placed into the balanced tubes. The centrifuge
can be operated manually or power operated. The balanced tubes can be rotated at different speeds. Due
to centrifugal force the suspended particles settle down at the bottom of the test tubes. The supernatant
can be discarded and the sediment contains the organelle (depending on the speed used to rotate the
centrifuge) .
The principle of centrifugation is based on the action of centrifugal force. Centrifugal force is the
imaginary radial force that appears to an observer to act outwards on a body moving in a curved path. The
real force is the centripetal force that constrains the body to move in a curved path. The centripetal force
on a body moving in a circle at a certain velocity is calculated as,
mv 2/r
Where m = mass of the body.
r = radius of the circle.
v = velocity.
Ultra-centrifuge is a centrifuge used to determine the molecular weight in high polymers and proteins. It
is rotated at very high speed.
The process of cell fractioning involves
a. The breaking of the cells by homogenization in a sucrose
buffer solution.
b. The homogenate is taken and subjected to low
centrifugation or low centrifugal force. This is done for a
short period so that the largest cell organelles like the
nucleus settles down or sediment as pellets.
c. After removing the sediment, the supernatant is now
subjected to higher centrifugal forces so as to gradually
remove the next heavier organelles.
d. The mitochondria are taken out followed by the endoplasmic
reticulum and membranes of the Golgi complex and
vacuoles. The last to form the sediment are the ribosomes.
e. To remove the ribosomes ultra-centrifugation is required.
f. The remaining supernatant consists of the cytoplasm and
other small substances too small to form sediments.
The cell is a microscopic unit that contains various cell organelles like, nucleus, mitochondria, Golgi
complex, lysosomes etc. If one needs to study a particular organelle, or to examine their contents or to
isolate an enzyme to study its activities, the organelle needs to be isolated. To isolate the required
organelle in a purified state and in bulk it is done by subjecting the cells to differential centrifugation. It
is known that we can separate the heavier particles from lighter ones by centrifugation. When the cell
extracts are centrifuged the heaviest organelle will be the first to settle down, followed by the next heavier
one and finally the lightest organelle or substances will settle down.
The separation of the cell organelles can be done as they vary in mass, surface and specific gravity and
settle down at different speeds when subjected to centrifugation. Depending on the speed the standard or
ultracentrifuges are used.
Cell fractioning is the process by means of which the different cell organelles are separated by
differential centrifugation. The various cell organelles vary in density, mass, surface and specific
gravity. When subjected to centrifugal force they tend to settle down at different speeds. They can then be
separated easily and later purified. The speed at which each organelle settles down at different and can be
expressed in terms of Svedberg units (S). The ribosome units are labeled as 80S or 70S.
The principle
Cell purification is achieved by centrifugation. Centrifugation is done by an apparatus called a
centrifuge. It has balanced tubes attached to the opposite ends of arms rotating rapidly about a central
point. The cell extract is poured into the test tubes and placed into the balanced tubes. The centrifuge
can be operated manually or power operated. The balanced tubes can be rotated at different speeds. Due
to centrifugal force the suspended particles settle down at the bottom of the test tubes. The supernatant
can be discarded and the sediment contains the organelle (depending on the speed used to rotate the
centrifuge) .
The principle of centrifugation is based on the action of centrifugal force. Centrifugal force is the
imaginary radial force that appears to an observer to act outwards on a body moving in a curved path. The
real force is the centripetal force that constrains the body to move in a curved path. The centripetal force
on a body moving in a circle at a certain velocity is calculated as,
mv 2/r
Where m = mass of the body.
r = radius of the circle.
v = velocity.
Ultra-centrifuge is a centrifuge used to determine the molecular weight in high polymers and proteins. It
is rotated at very high speed.
The process of cell fractioning involves
a. The breaking of the cells by homogenization in a sucrose
buffer solution.
b. The homogenate is taken and subjected to low
centrifugation or low centrifugal force. This is done for a
short period so that the largest cell organelles like the
nucleus settles down or sediment as pellets.
c. After removing the sediment, the supernatant is now
subjected to higher centrifugal forces so as to gradually
remove the next heavier organelles.
d. The mitochondria are taken out followed by the endoplasmic
reticulum and membranes of the Golgi complex and
vacuoles. The last to form the sediment are the ribosomes.
e. To remove the ribosomes ultra-centrifugation is required.
f. The remaining supernatant consists of the cytoplasm and
other small substances too small to form sediments.
