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Differential centrifugation technique
It is a type of preparative centrifugation. This method is based on the differences in the sedimentation rate of particles of different size and density. In differential centrifugation, the material (a tissue homogenate) to be separated in solution is centrifugally divided in to a number of fractions by the step wise increase of applied centrifugal field. The centrifugal field is determined by trial and error method so that the particular type of material sediments during predetermined time of centrifugation to sediment the particles in the form of pellet. The supernatant contains other materials which are unsedimented. At the end of each stage the pellet and supernatant are separated and the pellet is purified by washing. Initially, all particles of the homogenate are homogenously distributed through out the centrifuge tube. During centrifugation, particles move down the centrifuge tube at their respective sedimentation rates and start to form pellet at the bottom of the tube. Centrifugation can be continued till all the components are pelleted one by one by increasing the centrifugal field.
For example, the sub-cellular organelles (nucleus, mitochondria, lysosomes, microsomes) from a tissue liver homogenate can be isolated by applying this differential centrifugation techniques. The technique has the following steps:
a) Preparation of liver homogenate – 10% solution in 0.25 molar sucrose.
b) Centrifugation at 1000 g for 10 minutes.
c) Isolation of the pellet sedimented which is nucleus.
d) The supernatant decanted from step (c) is subjected to centrifugation at 3300 g for 10 minutes.
e) Isolation of the pellet sedimented which contains mitochondria.
f) The supernatant decanted from step (e) is subjected to centrifugation at 16300 g for 20 minutes.
g) Isolation of the pellet sedimented which contains lysosomes.
h) The supernatant decanted from step (g) is subjected to centrifugation at 105000 g for 60 minutes.
i) Isolation of the pellet sedimented which contains microsomes.
j) The supernatant obtained in the final step is the cell free cytosol.
The isolation of sub-cellular organelles is an essential procedure used in many biochemical research laboratories by using this differential centrifugation techniques. A schematic diagram of step-wise isolation sub-cellular organelles from a liver homogenate is given in Fig. 10.8.
Applications of analytical ultra-centrifuge: The analytical ultra-centrifuge has found many applications in fields of protein and nucleic acid chemistry. This gives information about (a) determination of molecular weight of biomolecules, (b) estimation of purity of macromolecules and (c) detection of conformational changes in macromolecules.
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