Cell fractionation

What is cell fractionation?

The process where cells are broken up (lysed) and the different organelles within the cells are separated out.

What conditions does the solution have to be and why?

• Cold- Reduce enzyme activity that could break down the organelle

• Buffered- So the pH doesn't fluctuate since a change in pH could effect organelles and enzymes (denatures proteins)

• Same water potential as the tissue/isotonic- To prevent organelles bursting or shrinking as a result of osmotic loss or gain of water.

Describe the stages of cell fractionation.

1) Homogenisation- this breaks open the cells, usually done either by vibrating the cells or grinding them up in a homogeniser (blender). This releases the organelles from the cells.

2) Filtration- the resulting fluid (homogenate) is filtered to remove any debris (whole cells or large bits of remaining tissue).

3) Ultracentrifugation- the fragments in the filtered homogenate are separated in a centrifuge at increasing speeds.

What happens during ultracentrifugation?

After spinning, the densest sediment form a pellet at the bottom of the tube while smaller, less dense components remain in suspension above (a portion called the supernatant).

During ultracentrifugation what component of the cell would form a pellet first?

The nucleus