cell fractionation

• state the steps of cell fractionation?

  step 1: homogenisation - cells are broken open, using a blender to release their contents.

  • cells must be prepared in a cold, isotonic, buffered solution

  step 2: filtration -

  • the tissue is then filtered to remove cell debris or any remaining cells.

  step 3: ultracentrifugation

  • filtered solution is spun at different speeds in a centrifuge therefore organelles are separated according to their densities.
  • material is centrifuged at a low speed first, pallet removed and supernatant respun at a medium speed then high speed.
• why should cells be prepared in a cold, isotonic, buffered, solution?

  • cold - to reduce enzyme activity, as when cells are broken, enzymes are released this could damage organelles.
  • isotonic - must be the same water potential to prevent osmosis as this could cause organelles to shrivel or burst.
  • buffered - cells are at constant PH, to prevent enzymes from being denatured.
• how are organelles separated when undergoing ultracentrifugation?

  by their density and size

  • largest/most dense organelles separate at low speeds (e.g. nuclei)
  • small/light organelles separate at high speeds (e.g. ribosomes)