Density Gradient Centrifugation Principle, Protocol, Uses

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Density Gradient Centrifugation

Density gradient centrifugation refers to an approach to separation between molecules, where the separation is determined by the density of the molecules when they travel through a gradient under the force of centrifugal.

Principle of Density gradient centrifugation

Density gradient centrifugation relies on the premise that molecules settle under the force of centrifugal forces until they are in contact with the same density in a medium identical to theirs. In this instance it is a medium that has the density gradient is utilized and needs to reduce density or increase it. Molecules within a sample travel through the medium while the sample rotates, creating the force of centrifugal. The heavier molecules begin to move towards bottom as they travel through an intensity gradient. The molecules are then suspended at a point which the density of particles is greater than that of the medium. This way molecules of different density are separated into different layers . These can be recovered through different methods.

Density gradient centrifugation Protocols

  1. A density gradient in an environment is made by gently placing the concentrations with lower levels over more concentrated ones in the centrifuge tube.
  2. The sample is placed on top of the gradient after which the tubes will be then placed inside an ultracentrifuge.
  3. The particles move through their gradients until they reach a level at which their density is comparable to with the denseness of medium around them.
  4. The particles are separated and separated, leaving the particles as separate units.

Uses of Density gradient centrifugation

  • Density gradient centrifugation is employed to purify huge quantities of biomolecules.
  • It is also used to cleanse diverse viruses that aid in their research.
  • This method can be utilized in both a separation method and also for determining the density of different particles.

Properties of density gradient

A perfect density gradient media includes the following properties:

  • Enough solubility to provide the necessary range of densities
  • Does not create solutions with high viscosity in the required density range.
  • Does not have to be hypoosmotic or hyperosmotic when the particles that are to be separated are highly osmotically sensitive
  • Solutions for the gradient must be adjusted to the pH and Ionic strengths that are compatible with the particles that are being separated.
  • Does not affect the biological activities of the sample
  • Nontoxic and not processed by cells
  • Do not disrupt assay protocols or cause a reaction with the tubes for centrifuge.
  • The property can be used to determine the level of concentration
  • Easy to get rid of the pure product
  • Autoclavable
  • Reasonable price

Examples of Density gradient centrifugation

  • This technique was employed during the famous study which showed DNA to be semi-conservative using various nitrogen isotopes.
  • Another instance is the use of this method to extraction of the microsomal portion from muscle homogenates. This is followed by segregation of membrane vesicles of different density.

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