MadSci Network: Cell Biology |
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
Try the links in the MadSci Library for more information on Cell Biology.