Cells

Cell fractionation

The process where cells are broken up and the organelles are separated out (We don’t want the organelles to be damaged)

Solution

Before cell fractionation, Cells being tested are placed in ice cold, buffered and isotonic solution (same water potential) of the same cell.

Isotonic

1. Same water potential as the tissue

2. To prevent osmosis

3. So organelles don’t burst or shrink

Buffered

1. Same pH

2. pH needs to be constant to prevent enzymes denaturing

Ice cold

1. Keep cell activities the same

2. By reducing enzyme activities (low temps)

3. So organelles aren’t digested

2 stages to Cell Fractionation

Homogenisation - Cells are broken up by homogeniser (blender) to release organelle from cell.

Fluid is filtered to remove complete cells and large pieces of debris.

Ultracentrifugation (Spinning Really Fast)

Liquid atop - Supernatant

Liquid at bottom - Pellet

Spin again if you want an organelle that’s smaller (e.g spin again if you want to get chloroplast, to get rid of nucleus) but spin faster.

Getting An Organelle

1. Tissue placed into isolated solution (buffered ice cold and isotonic)

2. Solution is placed in a homogeniser (blender) breaking apart cell and releasing organelles

3. The solution with organelles is filtered to remove complete cells and any large pieces of debris

4. The solution is placed in a tube and is centrifuged at low speeds.

5. Collect supernatant into a new tube and spin at higher speeds for to obtain smaller organelles.