Density gradient centrifugation

Centrifugation

1. DENSITY GRADIENT CENTRIFUGATION Amal George SBS MGU

2. Centrifugation Centrifugation is a process which involves the application of the centrifugal force for the sedimentation of heterogeneous mixtures with a centrifuge

3. Centrifugation

4. Centrifugation methods . . Differential Centrifugation Density Gradient Centrifugation Rate-Zonal Centrifugation Isopycnic Centrifugation

5. Density gradient Centrifugation A procedure for separating particles (such as viruses or ribosomes or molecules such as DNA )in which the sample is placed on a preformed gradient such as sucrose or caesium chloride . Upon centrifugation either by rate zonal or equilibrium procedures, the macromolecules are 'banded' in the gradient and can be collected as a pure fraction. Density gradient centrifugation are of two types: Rate zonal centrifugation Isopycnic centrifugation

6. Rate zonal centrifugation Isopycnic Centrifugation

7. 1. The sample is applied in a thin zone at the top of the centrifuge tube on a density gradient . 2. Under centrifugal force , the particles will begin sedimenting through the gradient in separate zones according to their size, shape, and density or the sedimentation coefficient(s ) 3.The run must be terminated before any of the separated particles reach the bottom of the tube. Rate zonal centrifugation ( Sedimentation velocity zone centrifugation )

8. This method is useful for separating particles which differ in size but not in density Extremely useful for the separation of proteins possessing nearly identical densities but differing only slightly in their molecular weights

9. Rate - zonal centrifugation is a centrifugation technique employed to effectively separate particles of different sizes. ... Once the centrifugation is over , fractions are collected

10. Isopycnic Centrifugation ( Sedimentation equilibrium centrifugation) In isopycnic separation, also called buoyant or equilibrium separation, particles are separated solely on the basis of their density. Particle size only affects the rate at which particles move until their density is the same as the surrounding gradient medium

11. overview

12. Applications Purification of particles separated by differential centrifugation Isolation of enzymes Purification and separation of viruses(pox viruses),bacteria… Purification and separation of proteins Separation of biomolecules Separation of RNA – DNA hybrids and ribosomal subunits Separation of antibodies and viruses