Differential centifugation :- Differential centifugation is separation of different components of a homogenate of sub-cellular particles by subjecting it to successively higher centrifugal force.
This bring about velocity sedimentation of various cell fragments at different speeds, depending upon their size and weight. The denser constituents settle down at lower speed, while the lighter components do so at higher speeds. The sediments or precipitate is called pellet while the remaining homogenate is called supernatant. The instrument used for creating centrifugal force is called centrifuge. It is motorised. Normal clinical centrifuge can rotate at a speed of upto 5000 revolutions per minute (rpm) or g.
(The rate of centrifugation is specified by acceleration applied to the sample, typically measured in revolutions per minute (rpm) or g.)
Centrifuges used in cell fractionation have high speeds of 50,000 - 100,000 rpm. They are called ultracenrifuges.
The rotor or rotating unit of ultracenrifuge is refrigerated and evacuated to prevent heating up and reduce friction. An ultracenrifuge operating at 80,000 rpm has a force of 5,00,000 times that of gravitational force.
Density gradient centrifugation :- In a centrifuge tube, a density gradient of sucrose or caesium chloride is established by means of special device. Maximum density occure at the bottom. It gradually decreases towards the top. Homogenate is poured over the sucrose solution. The tube is now centrifuged at high speed of 50,000 - 5,00,000 g or rpm for one or three hrs.
During this period different components of homogenate separate and form bands at different positions according to their sedimentation coefficeints or Svedberg units.
The latter depends upon the size and density of the components according to their Svedberg value is called isopyknic or equal density centrifugation.
It is useful in separating components of mitochondrial fraction ( mitochondria, lysosomes and microbodies) or fragments of SER from RER. At higher speeds even smaller molecules like t-RNA can be separated from larger molecules like m-RNA, DNA etc.
This bring about velocity sedimentation of various cell fragments at different speeds, depending upon their size and weight. The denser constituents settle down at lower speed, while the lighter components do so at higher speeds. The sediments or precipitate is called pellet while the remaining homogenate is called supernatant. The instrument used for creating centrifugal force is called centrifuge. It is motorised. Normal clinical centrifuge can rotate at a speed of upto 5000 revolutions per minute (rpm) or g.
(The rate of centrifugation is specified by acceleration applied to the sample, typically measured in revolutions per minute (rpm) or g.)
Centrifuges used in cell fractionation have high speeds of 50,000 - 100,000 rpm. They are called ultracenrifuges.
The rotor or rotating unit of ultracenrifuge is refrigerated and evacuated to prevent heating up and reduce friction. An ultracenrifuge operating at 80,000 rpm has a force of 5,00,000 times that of gravitational force.
Density gradient centrifugation :- In a centrifuge tube, a density gradient of sucrose or caesium chloride is established by means of special device. Maximum density occure at the bottom. It gradually decreases towards the top. Homogenate is poured over the sucrose solution. The tube is now centrifuged at high speed of 50,000 - 5,00,000 g or rpm for one or three hrs.
During this period different components of homogenate separate and form bands at different positions according to their sedimentation coefficeints or Svedberg units.
The latter depends upon the size and density of the components according to their Svedberg value is called isopyknic or equal density centrifugation.
It is useful in separating components of mitochondrial fraction ( mitochondria, lysosomes and microbodies) or fragments of SER from RER. At higher speeds even smaller molecules like t-RNA can be separated from larger molecules like m-RNA, DNA etc.
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