Density Gradient Centrifugation – Principle, Protocol, Uses

What is a Density Gradient Centrifugation?

Scientists apply density gradient centrifugation in laboratories to sort particles including cells, organelles, and biomolecules because of their density differences. During density gradient centrifugation a sample sits on top of a density gradient medium formed by substances such as sucrose or cesium chloride. During high-speed centrifuge spinning particles from the sample move to the location in the density gradient that matches their own density forming distinct bands.

Scientists frequently apply this method during biological and biochemical investigations. The method functions to cleanse large biomolecule amounts and viruses for research while isolating cellular components including DNA, RNA, proteins, and organelles. The method allows researchers to measure the density of various particles.

The advancement of molecular biology heavily depends on the development of density gradient centrifugation. Through its application scientists have achieved major breakthroughs including nucleic acid separation and macromolecular structure analysis. Researchers have come to rely on this technique which separates particles based on their density because of its importance in multiple scientific fields.

Principle of Density gradient centrifugation

During density gradient centrifugation particles in a mixture traverse through a medium until they encounter a position where their density equals the density of the medium. The method begins with a medium that has a known density gradient and during centrifugation particles travel through this medium. Particles move through the gradient until they reach an area where their density matches the medium’s density which then causes them to form separate bands based on density similarities.

Density gradient centrifugation Protocols

1. Prepare the Density Gradient– Select the proper substance such as sucrose or cesium chloride for gradient creation. Begin with the densest solution at the tube bottom and stack successively less dense solutions upward.
2. Load the Sample– Position the sample at the top of the density gradient with care to avoid disrupting the layered structure.
3. Centrifugation– Place the tube in the centrifuge. Centrifuge the tube using the settings that best match the size and density characteristics of the particles you are examining.
4. Fraction Collection– Once centrifugation is complete the particles will have moved to the location in the gradient where their density balances the density of the surrounding medium.
5. Collect fractions from the tube starting at the top and moving to the bottom to separate the components effectively.
6. Analysis– Study the collected fractions to identify the separated particles.

Uses of Density gradient centrifugation

• Researchers purify biomolecules which include proteins and nucleic acids for various applications.
• Isolation and purification of viruses for research
• Different cell types undergo separation according to their density differences.
• Scientists isolate cellular compartments such as mitochondria and nuclei for biological studies
• The evaluation of macromolecular uniformity together with their molecular weight distribution

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.