1. cell fractionation.pptx

Cell

1. Cell Fractionation Department of Zoology University of Narowal

2. Cell Fractionation Cell fractionation is a procedure for rupturing cells, separation and suspension of cell constituents in isotonic medium in order to study their structure, chemical composition and function.

3. Steps of Cell Fractionation Cell fractionation involves 3 steps: Extraction Homogenization Centrifugation

4. Extraction It is the first step toward isolating any sub-cellular structures . In order to maintain the biological activity of organelles and bio-molecules, they must be extracted in mild conditions called cell-free systems. For these, the cells or tissues are suspended in a solution of appropriate pH and salt content, usually isotonic sucrose (0.25 mol/L) solution. The sample are then kept cold to prevent enzymatic damage

5. Homogenization The suspended cells are then disrupted by the process of homogenization. It is usually done by: ( i ) Grinding ( ii) High Pressure (French Press or Nitrogen Bomb) (iii) Osmotic shock (iv) Sonication (ultrasonic vibrations) Grinding is done by pestle and mortar or potter homogenizer (a high-speed blender). The later consists of two cylinders separated by a narrow gap.

6. Centrifugation Last step of cell fractionation is the centrifugation. The separation (fractionation) of various components of the homogenate is carried out by a series of centrifugations in an instrument called preparative ultracentrifuge .

7. Differential centrifugation It is a process in which sequential centrifugation of a cell lysate at progressively increasing centrifugation force, isolating cellular components of decreasing size and density.

8. Equilibrium Density Gradient Centrifugation Equilibrium density gradient centrifugation is a procedure used to separate particles and mixed substances based on the difference in their  buoyant densities  ( density  = mass/volume).

9. Continue … The impure organelle fraction is layered on the top of a gradient solution, e.g., sucrose solution or glycerol solution. The solution is more concentrated (dense) at the bottom of the centrifuge tube and decreases in concentration gradually towards the top. The tube when centrifuged at high speed the various organelles migrate to an equilibrium position where their density is equal to the density of the medium.

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