APPLICATIONS OF CENTRIFUGATION

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Cell Fractionation

• Velocity sedimentation centrifugation separates particles ranging from coarse precipitates to sub cellular organelles.

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• In a typical cell fractionation , cells or tissues in solution are disrupted by gentle homogenization.

• This treatment ruptures the plasma membrane but leaves most of the organelles intact.

• The homogenate is then centrifuged; organelles such as nuclei, mitochondria, and lysosomes

differ in size and therefore sediment at different rates.

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• Differential centrifugation results in a rough fractionation of the cytoplasmic contents, which may be further purified by isopycnic (“same density”) centrifugation.• In this procedure, organelles of different buoyant densities (the result of different ratios of lipid and protein in each type of organelle) are separated on a density gradient. • By carefully removing material from each region of the

gradient and observing it with a microscope, the biochemist can establish the sedimentation position of each organelle

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• Particles of different density can be separated by isopycnic centrifugation. In isopycnic

centrifugation, a centrifuge tube is filled with a solution, the density of which increases

from top to bottom; a solute such as sucrose is dissolved at different concentrations to produce the density gradient.

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Analytical Measurements

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Density Gradient Centrifugation

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Zonal Centrifugation Prepared in Density Gradient

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• After centrifugation the contents of the tube are fractionated by drop collection from the bottom of the tube.

• In analytical centrifugation optical techniques are used to detect the molecules.

• The fractions obtained can be assayed by radioactivity, chemical tests, enzymatic activity etc or a combination of these.

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Uses

• Separation of enzymes, hormones, RNA-DNA hybrids, ribosomal subunits, subcellular organelles, for the analysis of size distribution of samples of polysomes and lipoprotein fractions.

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