Differential centrifugation

by

Minza Masunga

IPMB (I) 2010/2011

What is centrifugation

Centrifugation is a process that involves the use of centrifugal force(g-force) for the separation of mixtures.

Centrifuge machine

Differential centrifugation

Is the process of separatingorganelles /macromolecules fromcytosol and from each other by useof centrifugal force.Differential centrifugation used to

prepare sub cellular fractions orisolate specificorganelle/macromolecule.

Principle

In liquid media, different particles havedifferent density and shape.

So Centrifuge increases the effect ofgravity .

Many particles or cells in a liquidsuspension, given time, will eventuallysettle at the bottom of container due togravitational force.

Other particles are extremely small in size, will not separate at all in solution unless subjected to higher centrifugal force.

When a suspension is rotated at a certain speed or revolution per minute (RPM), centrifugal force causes the particles to move radially away from the axis of rotation.

The force on particles (compared togravity) is called Relative CentrifugalForce (RFC).

Example an RFC of 500xg indicatesthat the centrifugal force applied is500 €™ times greater than earthgravitational force.

During centrifugation each stepmore dense particles areseparated from less denseparticles, and successive speed ofcentrifugation increased until thetargeted pellet out.

Protocol

For study of subcelullar organelles, tissues or cells are first disrupted to release their internal contents.

The crude disrupted mixture is referred to as a homogenate.

During centrifugation of a cellhomogenate , larger particlessediments faster than small onesand this provides the basis forobtaining crude organelles bydifferential centrifugation.

A cell homogenate can be centrifugedat a series of progressively highergravitational forces and times togenerate pellets of partially purifiedorganelles.When a cell homogenate is centrifuged

at 1000xg for 10 minutes unbroken cellsand heavy nuclei pellet to the bottom ofthe tube.

The supernatant furthercentrifuged at 10,000xg for 20minutes pellet sub cellularorganelles of intermediatevelocities such as mitochondria ,lysosome and microbodies

Some of these sedimenting organellescan be obtained in a partial purity andare typical contaminated with otherparticles.

repeated washing the pellet byresuspending in isotonic buffer andrepelleting may result in removal ofcontaminants that are small in size.

Therefore, obtaining partiallypurified organelles by differentialcentrifugation serves as apreliminary step for furtherpurification using other types ofcentrifugation techniques e.g.density gradient separation.

Applications

In bioscience research to separate subcellularorganelles (mitochondria, nucleus,ribosomes),and isolation of macromolecules such as DNA,RNA, proteins, or lipids for further studies.

In Medicine-for diagnostic purposes

Example, purification of influenza virus for studieson fundamental chemical and physical propertiesof the virus leads to control and prevention ofinfluenza.

References

1. Wilson, K., Walker, J. (2006). Principles and Techniques of Biochemistry and Molecular biology, Cambridge university Press ,UK.

2. Berg, J.M., Tymoczko,L.J ., and Stryer, L. (2007). Biochemistry . W.H. Freeman and Co., New York.

3. Wikipedia.

Thank you!