Cell Fractionation/ Ultracentrifugation

Question 1

Definition of cell fractionation.

Answer 1

• Cell fractionation is the breaking down of cells to isolate different organelles so their structures/ functions can be studied.

Question 2

What is the first step of cell fractionation?

Answer 2

1.) Cells/ tissues are **broken open ** to release the contents/ organelles in an isotonic, buffered, cold solution.

Question 3

What are the 3 conditions of the solution that cells must be broken down in?

Answer 3

1.) Buffered.
2.) Isotonic.
3.) Cold

Question 4

Why is the solution that we break down cells into **buffered? **

Answer 4

• Maintains pH to prevent/ stop enzymes/ proteins from being denatured (enzymes/ proteins IN the organelles- damage to these proteins/ enzymes will damage the organelles themselves.)

() - extra info for clarification.

Question 5

Why is the solution that we break down cells into** isotonic? **

Answer 5

• Means that solution has same water potential to fluid in organelles.
• This wil prevent osmosis
• Stop/ prevent organelles bursting (lysis) or shrinking.

Question 6

Isotonic definition.

Answer 6

• Solution that has same concentration (of solute) as the solution it is being compared to ie. the solution in organelles.

Question 7

Why is the solution that we break down cells into **ice- cold? **

Answer 7

• When cell breaks open, enzymes are released (ie. from lysosomes), which could damage organelles.
• Prevents/ stops enzyme activity (which will prevent/ stop digestion of organelles.)

Question 8

What are the two main steps of cell fractionation?

Answer 8

1.) Homogenisation
2.) Ultracentrifugation.

Question 9

What is “homogenisation?”

Answer 9

• Breaking open cells/ releases organelles (cells being homogenised) in a blender known as a homogeniser.
• The cells are blended in a cold, isotonic, buffered solution.

Question 10

What do you do after you have homogenised cells/ released organelles?

Answer 10

• The solutio (also known as homogenate) is filtered to remove any unbroken cells/ large debris.

Question 11

What is the resultant fluid from the homogeniser called? What does this contain?

Answer 11

• Called homogenate.
• Contains organelles/ other fluids.

Question 12

How do organelles separate in a (ultra-) centrifuge?

Answer 12

• Organelles separate according to their densities (in an ultracentrifuge.)

Question 13

What do you do after you have filtered the homogenate?

Answer 13

• The filtered homogenate/ solution = placed in a (ultra) centrifuge and spun at different speeds.

Question 14

Describe differential (ultra) - centrifugation.

6 main stages

Answer 14

• Centrifuge spins at low speed (at first.)
• Solid pellet of the most dense organelles form at the bottom.
• The solution above the (solid) pellet is known as supernatant (contains other less dense organelles/ fluid.)
• The supernatant is removed - leaving behind the pellet of most dense organelles.
• The supernatant is spun at a higher speed to remove the next pellet of organelles (ie. the next density of organelles.)
• This process is repeating until a pellet of all organelles are separately formed.

Question 15

Order of organelles from most to least dense/ what pellet they would be collected in?

Answer 15

• Nuclei (collected in 1st pellet)
• Chloroplasts (if using plant tissue) - 2nd pellet.
• Mitochondria - 3rd pellet
• Lysosomes - 4th pellet
• ER - 5th pellet
• Ribosomes - 6th pellet.