Methods of studying cell fractionation

• what is the purpose of cell fractionation

  To obtain a sample of a given organelle for study in the lab
• give an overview of cell fractionation

  Cells are broken up (homogenisation) to release organelle, the debris is removed (filtration), and the organelles are separated using ultracentrifugation.
• describe how cells are homogenised

  The cell membrane is broken using a blender, releasing the organelles into the extraction solution.
• Describe and explain the composition of the extraction solution

  Ice-cold - Slows or stops enzyme activity to prevent digestion of organelles; Buffered - Maintains pH so that enzymes or proteins are not denatured; Same water potential as organelles-

  Prevents osmosis of water into/out of ORGANELLE so no bursting or shrinking of ORGANELLE (reject cell)
• explain why the homogenised cells need to be filtered

  to remove any cell debris or unbroken cells
• describe and explain how organelles can be separated using ultracentrifugation

  The filtrate is placed in a test tube and spun at the lowest speed. The largest/heaviest/most dense organelles form a thick sediment at the bottom of the tube called a pellet. The supernatant, which is the fluid where all the other organelles are suspended, is poured off, and spun again at a higher speed. The next largest organelles form the pellet. This is repeated at progressively higher speeds until all the organelles are separated.
• give the order that cell organelles will appear in the pellet at successively higher speeds

  Nucleas, mitochondria and chloroplasts, lysosomes, ribosomes
• Explain how the organelles are separated by ultracentrifugation

  The organelles are separated on their mass, size and density. The largest/most dense/most massive are in the first pellet when a low speed spin was used