Fractionation And Ultracentrifigation

Question 1

What is cell fractionation ?

Answer 1

The process of breaking up different cells and separation the different organelles out for study.

Question 2

How do you prepare for cell fractionation ?

Answer 2

Cells are placed in an isotonic solution which is:
Cold - reduces enzymes activity so that the cells do not break down. Also prevents auto Lydia.
Isotonic- Has the same water potential as the cell so that osmotic Lysis or shrinking of the cell does not occur.
Has a buffer- Maintains the correct Ph so that enzymes in the cell are not denatured. Prevents damage to enzymes and proteins.

Question 3

What are the 2 stages of cell fractionation ?

Answer 3

Homogenation and ultracentrifugation.

Question 4

Give a detailed description of how homogenisation occurs.

Answer 4

A sample tissue is broken up to release organelles using a blender or homogeniser. The resultant fluid is then filtered to remove any complete cells of debris.

Question 5

Explain Centrifugation.

Answer 5

A test tube filled with the homogenate is placed in the Centriguge machine and spun at a low speed. Centrifugal force is created, forcing the heaviest organelle to the bottom of the test tube.
A pellet of that organelle is left at the bottom while supernatant is left at the top.
The supernatant is then spun again at a higher speed to remove the next heaviest organelle.

Question 6

How is the supernatant separated from organelle pellet ?

Answer 6

The pellet sticks to the bottom of the cell while the supernatant can be poured into another test tube.

Question 7

List the organelles in the order that they are separated.

Answer 7

Nuclei
Lysosomes and Mitochondria
Membranes - Smooth endoplasmic reticulum and Rough endoplasmic reticulum.
Ribosomes.

Question 8

What speed and g force must the nuclei be spun at ?

Answer 8

10 minutes, low speed , 500-600 G force.

Question 9

What speed and g force must the Lysosomes and Mitochondria be spun at ?

Answer 9

Moderate speed, 15 minutes, 10000-20000 G force.

Question 10

What speed and g force must the membranes be spun at ?

Answer 10

High speed - 30 minutes.

Question 11

What speed and g force must the ribosomes be spun at ?

Answer 11

Very high speed for 45-60 minutes at 100000 G force.

Question 12

What does homogenise mean ?

Answer 12

Break up

Question 13

What is the supernatant ?

Answer 13

Remaining organelles suspended in liquid.

Question 14

What organelles may not be fully separated and could be separated in the fractions ?

Answer 14

Golgi body and endoplasmic reticulums.

Question 15

What is autolysis ?

Answer 15

The destruction of cells or tissues by their own enzymes.

Question 16

What property of cells allows them to separate out in different fractions ??

Answer 16

Varying masses and densities.

Question 17

If the cell preparation had been contaminated with bacteria, why might it have been difficult to separate the Mitochondria from the bacteria ?

Answer 17

They have similar masses and densities.

Question 18

If a cell has burst , what does this mean about water potential ??

Answer 18

Water potential outside the cell was higher and water has therefore moved into the cell leading to osmotic lysis.

Question 19

Explain in detail how you would separate out Mitochondria.

Answer 19

Disturb/ damage cells in blender to separate out organelles. 
Cold,isotonic,buffer. 
Filter homogenate. 
Spin in centrifuge - nucleus. 
Spin again - mitochondria.