Density gradient centrifugation,types, advantages and disadvantages

Density gradient centrifugation,types, advantages and disadvantages

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  Introduction • What iscentrifugation? • Centrifugation is a technique used for the separation of particles from a solution according to their size, shape, density, viscosity of the medium and rotor speed. The particles are suspended in a liquid medium and placed in a centrifuge tube. The tube is then placed in a rotor and spun at a define speed.
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  Types of centrifugation •There are three types of centrifugation:- • 1. Density gradient centrifugation • 2. Differential centrifugation • 3. Ultra centrifugation
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  Density gradient centrifugation •Definition: A method where the components of a sample are separated on the basis of their density, in a dense medium or density gradient, in a centrifuge, according to the centrifugal force they experience. • Principle :- The principle is based on a decreasing density of the suspending solution and migration of the targets to the equilibrate portion of the sample tube during centrifugation.
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  Generation of densitygradients
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  Classification • Density gradientcentrifugation is classified intro 2 types:- • 1. Rate zonal centrifugation:- Rate-zonal centrifugation is a centrifugation technique employed to effectively separate particles of different sizes. • 2. Isopycnic zonal centrifugation:- A method where the components of a sample (e.g. DNA) are separated on the basis of their density in a centrifuge according to the centrifugal force they experience.
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  1. Rate zonalcentrifugation • Rate-zonal centrifugation is a centrifugation technique employed to effectively separate particles of different sizes. • The tube is first filled with different concentrations of sucrose or another solute establishing layers with different densities and viscosities, forming a density gradient, within which the particles to be separated are added. • The larger particles will be able to travel to the bottom layer because they are more massive. The greater mass allows the particles to travel through layers with a greater viscosity, while the smaller particles will remain at the top, as they lack the mass to travel through the more viscous layers. • Once the centrifugation is over, fractions are collected.
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  Application
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  Isopycnic Centrifugation • IsopycnicCentrifugation • In isopycnic centrifugation, using a continuous gradient, a fairly homogeneous population of organelles will ‘band’ in the gradient at their actual buoyant density over several hours. • This method of separation is independent of time, and relies solely on the actual buoyant density of the particle. Banded organelles or particles can be recovered from the gradient by subjecting it to fractionation (either by punching a hole in the bottom of the tube and draining it, or by taking specific volumes of fractions from the top of the gradient). • If bands are visible, they may be removed individually with a syringe with a long needle.
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  Rate zonal centrifugationVs Isopycnic Centrifugation
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  Applications of densitygradient centrifugation
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  Thank you