2.2.6 Cell Fractionation & Ultracentrifugation

• Biologists and other scientific researchers may sometimes want to study one specific organelle
• This may be to:
  • Look at the appearance of the specific organelle under an electron microscope
  • Conduct research on the specific organelle (for example, to learn more about its function)

• To do this, a pure sample is needed (containing only the specific organelle being studied)
• The process of separating cell organelles from each other is known as cell fractionation
• This process involves breaking up a suitable sample of tissue and then centrifuging the mixture at different speeds
• Cell fractionation can be split into three stages:
  • Homogenisation
  • Filtration
  • Ultracentrifugation

Homogenisation

• Homogenisation is the biological term used to describe the breaking up of cells
• The sample of tissue (containing the cells to be broken up) must first be placed in a cold, isotonic buffer solution
• The solution must be:
  • Ice-cold to reduce the activity of enzymes that break down organelles
  • Isotonic (it must have the same water potential as the cells being broken up) to prevent water from moving into the organelles via osmosis, which would cause them to expand and eventually damage them
  • Buffered (have a buffer solution added) to prevent organelle proteins, including enzymes, from becoming denatured

• The tissue-containing solution is then homogenised using a homogeniser
  • This is a blender-like machine that grinds the cells up (the cells can also be vibrated until they break up)

•  This breaks the plasma membrane of the cells and releases the organelles into a solution called the homogenate

Filtration

• The homogenate (containing the homogenised cells) is then filtered through a gauze
  • This is to separate out any large cell debris or tissue debris that were not broken up
  • The organelles are all much smaller than the debris and are not filtered out (they pass through the gauze)
  • This leaves a solution (known as the filtrate) that contains a mixture of organelles

Ultracentrifugation

• The filtrate is placed into a tube and the tube is placed in a centrifuge
  • A centrifuge is a machine that separates materials by spinning

• The filtrate is first spun at a low speed
  • This causes the largest, heaviest organelles (such as the nuclei) to settle at the bottom of the tube, where they form a thick sediment known as a pellet
  • The rest of the organelles stay suspended in the solution above the pellet
  • This solution is known as the supernatant

• The supernatant is drained off and placed into another tube, which is spun at a higher speed
  • Once again, this causes the heavier organelles (such as the mitochondria) to settle at the bottom of the tube, forming a new pellet and leaving a new supernatant

• The new supernatant is drained off and placed into another tube, which is spun at an even higher speed
• This process is repeated at increasing speeds until all the different types of organelle present are separated out (or just until the desired organelle is separated out)
• Each new pellet formed contains a lighter organelle than the previous pellet
• The order of mass of these organelles (from heaviest to lightest) is usually:
  • Nuclei
  • Chloroplasts (if carrying out cell fractionation of plant tissue)
  • Mitochondria
  • Lysosomes
  • Endoplasmic reticulum
  • Ribosomes

The three stages of cell fractionation (homogenisation, filtration and ultracentrifugation) for separating organelles