2. Centrifugation
Centrifugation is a process which involves the application of the
centrifugal force for the sedimentation of heterogeneous mixtures with
a centrifuge
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
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.
11. 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
14. 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
