MadSci Network: Cell Biology
Query:

Re: What is density gradient centrifugation?

Date: Sun Dec 3 23:17:44 2000
Posted By: Elsa Lee, Grad student, Molecular Cell Biology, University of California, Berkeley
Area of science: Cell Biology
ID: 975087218.Cb
Message:

Dear Anil,

As you may know cells contain a variety of organelles. Density gradient 
centrifugation is a method which scientists use to separate intact 
organelles from a cell. Each type of organelles in a cell has an specific 
density and based on that density you can separate a certain type of 
organelle from the others. 

First, cells are gently lysed with a mild detergent to release all their 
cellular components. Next, this extract ( usually called a cell lysate) is 
filtered to remove any broken membranes. The solution is then added to a 
solution that is very dense like cesium cholride, sucrose, or  glycerol.  
The tube is then centrifuged at very high speeds for several hours. When 
you centrifuge at high speed, the dense solution you added to the cell 
lysate, creates a gradient in the tube. Heavier items will mirgrate to the 
bottom of the tube whereas lighter items will go to the top. During this 
time, each organelle wiil migrate to its appropriate equilibrium density 
and stay there. For example, mitochondria have a density of 1.18 
gram/cubic centimeter. While another type of organelle, the  peroxiosome 
have a density of 1.23 gram/cubic centimeters. When you look 
at the tube you will see many  bands at different levels ( mitochondria 
will be higher than the peroxiosome). The lower the band ( near the bottom 
of the tube) the more dense the particles. Now you can isolate your 
organelle of interest. You can look under a microscope to check if your 
organelles are intact. 

Once you have isolated your organelle of interest, you can now perform 
experiments to study function. With intact and live mitchondria you are 
now able to extract information how they behave inside a cell.

To learn more about density centrifugation I suggest you look in a 
textbook by Lodish et al., called: "Molecular Cell  Biology. " 
 
If you have more specific questions regarding your project, please feel 
free to email me.

Have fun with science!
Elsa



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