differential centrifugation

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1. DIFFRENTIAL CENTRIFUGATION Done by M.Rakshmitha 5 th semester ,Biotechnology

2. Differential centrifugation is a type of ultra centrifugation technique which comes under preparative ultra centrifugation. This technique is a common procedure in microbiology & cytology. The main essence of this centrifugation is to separate certain cell organells from a cell to study & analyse the specific parts of the cell. In this process, a tissue sample is first homogenised to break the cell membrane and mix up the cellular contents .

3. Mixture of homogenate particles can be separated by centrifugation on the basis of their densities or size. This can be achieved either by knowing the time required for their complete sedimentation in a fixed centrifugal field or the extent of their sedimentation after a given time in a fixed centrifugal field. In the same manner we can vary the value of centrifugal field applied for the each intracellular organelle and again find out the time required for each of them to sediment completely under various centrifugal fields. For example- upon subjecting a homogenate mixture of cell organelles for centrifugation for 15minutes at 1000g, the cell organelles like mitochondria and lysosomes will pellet out.

4. The best stratagy that we can adapt now to separate the tissue homogenate into various cell organelles is to centrifugally divide the homogenate into number of fractions by increasing the centrifugal field at each step. Centrifugal field should be choosed in a manner by knowing that a particular organelle will sediment at a known time to give a pellet. The pellet and supernatent will be seperated in each step and the supernatent is recentrifugated to sediment another light intra cellular organells .

5. Every pellet has to be washed several times because the pellet obtained is never pure. That means the pellet will contain contamination from other particles contained in the homogenate apart form the desired.

6. Advantages and Disadvantages of Differential centrifugation For certain experiments which require absolute purity density gradients methods can be used