2: CELLS - CELL FRACTIONATION

Question 1

What is cell fractionation?

Answer 1

a process used to separate organelles

Question 2

What are the 3 steps involved in cell fractionation?

Answer 2

• homogenisation (breaking up the cells)
• filtration (removing debris)
• ultracentrifugation (separating the organelles)

Question 3

Homogenisation

Answer 3

• can be done in several ways (eg: vibrating the cells/grinding up the cells in a homogeniser)
• plasma membrane broken up and organelles released into solution

Question 4

What are the conditions required for homogenisation?

Answer 4

• solution must be kept ice-cold to reduce activity of lytic enzymes which break down organelles
• solution should also be isotonic (same conc. of chemicals as the cells being broken down) to prevent damage to organelles through osmosis
• a buffer solution should be added to maintain pH

Question 5

Filtration

Answer 5

• homogenised solution is filtered through a gauze to separate any large cell debris/tissue debris (eg: connective tissue) from the organelles
• organelles pass through gauze as they are much smaller than the debris

Question 6

Ultracentrifugation

Answer 6

• homogenate poured into tube and put into centrifuge which is then spun at a low speed
• the heaviest organelles form a thick sediment (pellet) at the bottom and the rest of the organelles stay suspended in the fluid above (supernatant)
• supernatant is poured into another tube and the process is repeated at higher and higher speeds until all the organelles are separated out

Question 7

In what order are organelles separated out during ultracentrifugation?

Answer 7

• order of mass:
  
  • nucleus
    (- chloroplast)
  • mitochondria
  • lysosomes
  • endoplasmic reticulum
  • ribosomes