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DESCRIPTION
Two types of centrifugation are possible 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 organelles can be investigated in detail Two types of centrifugation are possible Differential Centrifugation Density gradient Centrifugation Isolated organelles are separated on the basis of their weight Isolated organelles are separated on the basis of their density Solutions of increasing density, such as sucrose solutions, are layered into a test tube with the most concentrated solution at the bottom of the tube The suspension of isolated organelles is spun for different combinations of speed and time The suspension of isolated organelles is pipetted onto the top of the most dilute solution The lowest speed and shortest time separates out the heaviest organelles with high speeds and longer times separating the lightest organelles As the tubes are spun, the organelles collect in the layer which corresponds to their own densityTRANSCRIPT
© SSER Ltd.
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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