Describe and explain how cell fractionation and ultracentrifugation can be used to isolate mitochondria from a suspension of animal cells.

It is possible to separate out the different organelles of a cell by spinning a homogenised solution of them at different speeds using an ultracentrifuge. The cells must first be placed in a cold, isotonic buffer solution to prevent damage to the organelles: the low temperature reduces enzyme activity that might break them down, an isotonic solution will prevent bursting and shrinking, and a buffer maintains the pH to prevent proteins denaturing. The cells should then be homogenised to break them open, releasing the mitochondria and other organelles from within the cell membrane. The resulting mixture must then be filtered to remove debris & remaining whole cells. The mixture must then be centrifuged (starting off at a low speed, around 1000 g) to separate nuclei, large cell fragments, and or other heavy organelles. The resulting supernatant (remaining solution) must be re-spun at higher speed than used previously so that the mitochondria form a solid pellet at the bottom. When answering questions like this in an exam, it is important to note that the question refers to animal cells - references to cell walls and other organelles specific to plant cells will reduce the number of marks available to you!