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The technique of ultracentrifugation is used to extract and isolate pure samples of cell organelles for study - this is one method of cell fractionation

Once isolated, the chemistry and functioning of the organellescan be investigated in detail

Two types of centrifugation are possible

DifferentialCentrifugation

Density gradientCentrifugation

Isolated organelles are separatedon the basis of their weight

The suspension of isolated organelles is spun for different combinations of speed and time

The lowest speed and shortest timeseparates out the heaviest organelles

with high speeds and longer timesseparating the lightest organelles

Isolated organelles are separatedon the basis of their density

Solutions of increasing density, suchas sucrose solutions, are layered into

a test tube with the most concentratedsolution at the bottom of the tube

The suspension of isolated organelles ispipetted onto the top of the most dilute solution

As the tubes are spun, the organellescollect in the layer which corresponds

to their own density

Ultracentrifugation is a technique used for the isolationof pure samples of cell organelles

The isotonic solution preventsorganelles from shrivellingor bursting due to osmotic

effects and the low temperatureprevents any cellular chemical

activity from taking placeThe internal organelles of the cell are released into a

suspension called the homogenate

The homogenate is transferredinto centrifuge tubes

This first sample is then centrifuged at low speedfor a short period of time

Chopped liver tissue is homogenised in an isotonic salt solution kept at 2°C

The heaviest organelles(nuclei) collect at the

bottom of the tube

The supernatant from the first sample istransferred to a second centrifuge tube

The sediment or pellet from the first sampleis removed and examined

This second sample is then centrifuged at moderate speedfor a longer period of time

Sample 2

The moderately heavy organelles (mitochondria)

collect at the bottomof the tube

The supernatant from the second sample istransferred to a third centrifuge tube

The sediment or pellet from the second sampleis removed and examined

This third sample is then centrifuged at high speed forAn even longer period of time

Sample 3

The light organelles(rough endoplasmic reticulum) collect at the bottom of the tube

Centrifuge at low speedfor 10 minutes

Centrifuge at moderate speed for 15 minutes

Centrifuge at high speedfor 30 minutes

The suspension of homogenised tissue,in a solution of density 1.0 g cm-3 ispipetted onto the top of the most

dilute sucrose solution

Solutions of decreasing density ofsucrose are placed into centrifuge tubes

The density of the sucrose solutionis approximately 1.20 at the bottom

and 1.00 at the top

decreasingdensity

The tubes are spun in a centrifuge

As the tubes are centrifuged, the organelles move towards the bottom of the tube until they reach a level at which their density

corresponds to that of the surrounding sucrose solution

Fractions of the contentsof the tube are removedthrough a puncture in

the bottom of the centrifuge tube

The samples of organelles are distributed in the differenttubes according to their density

Different organelles(which have differentdensities) ‘band’ in the sucrose of their

own density

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