Cell Fractionation Flashcards

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1
Q

What are the steps of cell fractionation?

A
  1. Homogenisation
  2. Filtration
  3. Ultracentrifugation
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2
Q

Describe homogenisation

A
  • break down cells to release organelles by vibrating cells or grinding cells in a blender
  • conditions of solution: ice cold, isotonic, buffered
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3
Q

What are the 2 methods to break plasma membrane and release organelles?

A
  1. Vibrating cells
  2. Grinding cells in a blender
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4
Q

What are the 3 conditions of the homogenised solution?

A
  1. Ice cold
  2. Isotonic
  3. Buffered
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5
Q

Why should the homogenised solution be ice cold?

A

Slow down enzyme activity to prevent denaturing

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6
Q

Why should homogenised solution be isotonic?

A

Water potential of the solution should be the same as the cells that have been broken down to prevent osmosis and therefore damage

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7
Q

Why should the homogenised solution be buffered?

A

Ensure pH stays constant so proteins are not denatured

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8
Q

Describe the process of filtration

A
  • homogenised solution filtered through a gauze to remove tissue and cell debris
  • organelles are much smaller than debris so aren’t filtered out
  • leaves filtrate containing mixture of organelles
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9
Q

Describe the process of ultracentrifugation

A
  • filtrate poured into tube and put into centrifuge and spun at low speed
  • supernatant drained off and poured into another tube and spun at higher speed
  • process repeated at increasing speeds until all organelles are separated
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10
Q

What are pellets?

A

The heaviest organelle settled at the bottom of the tube

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11
Q

What is the mass order from pellet 1-4?

A
  1. Nuclei
  2. Mitochondria and chloroplast
  3. Lysosomes
  4. Endoplasmic reticulum and ribosomes
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