Cell fractionation process

 

Before cell fractionation: Place tissue in isotonic buffered solution -low temperature reduces enzyme activity, which may break down the organelles -the isotonic solution prevents organelles bursting or shrinking as a result of osmotic gain or loss of water. The isotonic solution has the same water potential as the origional tissue. -the solution is buffered to maintain a constant pH.

Stage 1. Homogenation Cells are broken up by a homogeniser (blender), which releases the organelles from the cells. The resultant fluid (homogenate) is filtered to remove any complete cells and large debris. The filtrate (filtered homogenate) contains the organelles.

Stage 2. Ultracentrifugation A machine called a centrifuge separates the fragments in the filtrate into organelles. The filtrate is placed in a tube and the tube is placed into a centrifuge. The centrifuge spins the filtrate at a slow speed, which causes the heaviest organelles to sediment on the bottom- forms a pellet. The heavy organelles in the pellet are nuclei. The fluid (supernatant) is poured into another tube and spun at a faster speed. The next heaviest organelles are sedimented, which are mitochondria. This process is continued to produce pellets of organelles of varying sizes.