Centrifugation.pdf

Centrifugation  Centrifugal Motion ◼ Centrifugal acceleration = rω2 ◼ ω is the angular velocity in rad/s ◼ r is the radius of rotation ◼ Centrifugal force = mrω2 ◼ m is the mass of the particle ◼ The sedimentation coefficient, S, is ratio of a velocity to the centrifugal...

Centrifugation  Centrifugal Motion ◼ Centrifugal acceleration = rω2 ◼ ω is the angular velocity in rad/s ◼ r is the radius of rotation ◼ Centrifugal force = mrω2 ◼ m is the mass of the particle ◼ The sedimentation coefficient, S, is ratio of a velocity to the centrifugal acceleration (unit is second). S of 1X10-13 second is defined as one Svedberg unit (1S = 10-13 second). Velocity  To separate two miscible substances To analyze the hydrodynamic properties of macromolecules Purification of mammalian cells Fractionation of sub-cellular organelles (including membranes / membrane fractions) Fractionation of membrane vesicles Separating impurities from water Removing fat from milk to produce skimmed milk Separating particles from an air-flow using cyclonic separation The clarification and stabilization of wine Separation of urine components and blood components in forensic and research laboratories Aids in separation of proteins using purification techniques such as salting out, e.g. ammonium sulfate precipitation.  Cell fractionation Biologists need to study certain organelles from a cell (the mitochondria of a human cell or the chloroplasts of a plant cell, for example. Isolating these organelles involves a variety of procedures collectively called cell fractionation. What we’ve learned so far using this technique : 1.Mechanism of protein synthesis 2.DNA replication and transcription 3.RNA splicing 4.Muscle contraction 5.Microtubule assembly 6.Vesicular transport in the secretory pathway 7.Importance of mitachondria and chloroplasts in energy interconversions. Cell fractionation methods Involve the homogenization or destruction of cell boundaries by different mechanical or chemical procedures, followed by the separation of the subcellular fractions according to mass, surface, and specific gravity Steps of subcellular fractionation 1. Homogenization 2. Differential centrifugation 3. Further separation and purification by density gradient centrifugation 4. Collection of fractions 5. Analysis of fractions Homogenization or Cell Disruption Chemical : alkali, organic solvents, detergents Enzymatic : lysozyme , chitinase Physical : osmotic shock, freeze/thaw Mechanical : sonication , homogenization, French press