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Ultracentrifugation Assignment

Ultracentrifugation uses very high centrifugal forces, up to 1 million g, to separate biological particles suspended in liquid based on differences in weight. It is a key technique in biochemical research for analyzing cells, organelles, and molecules like proteins. During ultracentrifugation, biological structures sediment at different rates depending on their density, mass, and other factors. This allows separation of particles into pellet and supernatant fractions. Differential centrifugation employs increasing centrifugal forces to separate a tissue homogenate into fractions of organelles and other structures based on their size and density.

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801 views3 pages

Ultracentrifugation Assignment

Ultracentrifugation uses very high centrifugal forces, up to 1 million g, to separate biological particles suspended in liquid based on differences in weight. It is a key technique in biochemical research for analyzing cells, organelles, and molecules like proteins. During ultracentrifugation, biological structures sediment at different rates depending on their density, mass, and other factors. This allows separation of particles into pellet and supernatant fractions. Differential centrifugation employs increasing centrifugal forces to separate a tissue homogenate into fractions of organelles and other structures based on their size and density.

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Stuti Srivastav
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© © All Rights Reserved
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ULTRACENTRIFUGATION

Biological centrifugation is the process which uses centrifugal force to separate and purify mixtures of
biological particles in a liquid medium. It is a key technique for analysing cells, subcellular fractions,
supramolecuar complexes and isolated molecules such as proteins or nucleic acids.

Ultracentrifugation makes use of a centrifuge optimized for spinning rotor at very high speeds, capable of
generating accelerations as high as 1,000,000 g. It is an important tool in biochemical research, which
through rapid spinning imposes a high centrifugal force on suspended particles and causes separation of
such matter on the basis of differences in weight.

Basic principle :

Biological structures exhibit a drastic increase in sedimentation when they undergo acceleration in a
centrifugal field.

• the more dense the biological structure, the faster it sediments in a centrifugal field.
• the more massive the particle, the faster it moves in this field.
• the denser the biological buffer system is, the slower the particle will move in the centrifugal
field.
• the greater the frictional coefficient is, the slower a particle will move
• the greater the centrifugal force, the faster the particle sediments.
• sedimentation rate will be zero when the density of the particle and the surrounding medium are
equal.

Ultracentrifugation has decisively advanced in detailed biochemical analysis of subcellular structures and
isolated biomolecules.

Fig. Schematic diagram of an ultracentrifuge

TYPES OF CENTRIFUGES :
Many different types of centrifuges are commercially available including :

1. Large-capacity low-speed preparative centrifuges.


2. Refrigerated high-speed preparative centrifuges.
3. Analytical ultracentrifuges.
4. Preparative ultracentrifuges.
5. Large-scale clinical centrifuges.
6. Small-scale laboratory microfuges.

DIFFERENTIAL CENTRIFUGATION :

Isolation of large cellular structures, the nuclear fraction, mitochondria, chloroplasts or large protein
precipitates can be achieved by conventional high speed refrigerated centrifugation. Differential
centrifugation is based upon the differences in the sedimentation rate of biological particles of different
size and density.

Crude tissue homogenates containing organelles, membrane vesicles and other structural fragments are
divided into different fractions by the stepwise increase of the applied centrifugal field.
Following the sedimentation of the largest particle of the homogenate, various biological structures or
aggregates are separated into pellet and supernatant fractions, depending on the speed the time of
individual centrifugation steps and the density and relative size of particles.

To increase the membrane structures and protein aggregates released, cellular debris pellets are often
rehomogenised several times and then recentrifuged.

Initially, all particles of the homogenate are evenly distributed throughout the centrifugation tube and
then move down the tube at their respective sedimentation rates during centrifugation. Largest class of
particles forms a pellet at the bottom of the tube, leaving smaller sized structures within the supernatant.
However, initially the smaller particles also become entrapped in the pellet causing a certain degree of
contamination.

After each differential centrifugation step, supernatant and pellet are carefully separated from each other.
Pellets are washed several times with resuspension buffers and recentrifuged to minimise cross-
contamination.

One disadvantage is that repeated washing steps may reduce yield of final pellet fraction, specially if the
starting material is limited.
Resulting supernatant fractions are centrifuged at higher speeds and for longer time to separate medium-
sized and small-sized particles. Crude differential centrifugation techniques may be employed to isolate
mitochondria and microsomes.

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