A4 Cellular fractionation

Subcellular fractionation is the breaking open of a cell (e.g. by homogenization) and the separation of the various organelles from one another by centrifugation. 

This procedure uses a gradient of a dense solution (e.g. sucrose solution) to separate out subcellular organelles on the basis of their density. An ultracentrifuge is used to sediment the organelles to an equilibrium position in the gradient where their density is equal to that of the sucrose. 

A convenient way of determining the purity of an organelle preparation is to measure the activity of a marker enzyme in the various subcellular fractions. A marker enzyme is one that is found within only one particular compartment of the cell. 

Individual cells can be identified using a flow cytometer. Antibodies, coupled to fluorescent compounds, that bind to molecules on the surface of particular types of cells can be used to separate cells from each other in a fluorescent-activated cell sorter. 

Instant Notes in Biochemistry 2nd Edition, B.D. Hames N.M. Hooper, (c) 2000 BIOS Scientific Publishers Ltd, Oxford. 

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