Centrifugation

Question 1

What are the different components of a centrifuge?

Answer 1

• refrigeration unit
• rotor
• motor
• armoured bowl
• armoured locking lid

Question 2

What is the role of the rotor?

Answer 2

The rotor drives the centrifuge shaft

Question 3

How can we convert N to relative centirfugal force?

Answer 3

Divide the force value by gravity (9.81ms⁻¹ )

can also use a nonogram

Question 4

Which sized particle will sediment faster and why?

Answer 4

The bigger the particle the faster it will sediment as the centrifugal force will be bigger

Question 5

What is the acceleration of a particle dependent on?

Answer 5

Acceleration is affected by

• distance from axis (r)
• spin rate squared (w²)

therefore acceleration = w² x r
so F = m x w²r

Question 6

What does the rate of sedimentation depend on?

Answer 6

• centrifugal force
• buoyant density (less dense particles float)
• frictional drag

Question 7

What is the Svedberg unit?

Answer 7

The measure of a particles size based on its sedimentation rate (how long it takes to form pellet)
not additive

Question 8

How is rpm converted to angular velocity?

Answer 8

Divide by 60
Multiply by 2n
=> w = 2n x rpm/60

Question 9

How is a centrifuge used to separate cells?

Answer 9

Mammalian cells are delicate can be separated using shear forces
at 50-100g
for 5-10 minutes

Question 10

Outline the process of isopycnic centrifugation

Answer 10

1. Centirufge at 100,000g for 16hrs
2. RNA denser than DNA so forms pellet first
3. DNA forms a band where CsCl density is equal to DNA
   density

Question 11

How is a centrifuge calibrated?

Answer 11

Balanced by eye

Question 12

What is a microsome?

Answer 12

A fragment of ER and attached ribosomes obtained by centrifuging homogenised cells

Question 13

What is subcellular fractionation?

Answer 13

Breaking open mammalian cells using mechanical stress in a homogeniser
(aka Ultracentrifugation)

Question 14

What are the 2 forms of rotor in a centrifuge?

Answer 14

1. Swing out rotor:
   
   • Tubes swing out in line with G force (horizontally)
2. Fixed angle rotor
   
   • Tubes held at an angle, so pellet forms at side of tube
     (not bottom)

Question 15

Which is the densest organelle in a cell?

Answer 15

The nucleus - forms pellet first

Question 16

Why is caesium chloride used in isopycninc centrifugation?

Answer 16

Cs is a heavy metal
CsCl is a very soluble and dense copound
When spun in a centrifuge spontaneously forms a density gradient

Question 17

Give an equation used to calculate the centrifugal force

Answer 17

Force = Mass x Acceleration

Question 18

How can nuclear fractionation be further purified?

Answer 18

Via pellet ‘washing’

1. Re-suspend pellet in buffer + sucrose
2. Re centrifuge
3. Density gradient centrifugation

Question 19

What units are used in calculating the force?

Answer 19

Acceleration - ms⁻²
Force - N (newtons)
Spin - radiansˢ⁻¹ (angular velocity)

Question 20

What is the role of the armoured lid?

Answer 20

Prevents the centrifuge opening whilst in use - preventing injury

Question 21

What is the purpose of the buffer and sucrose in nuclear fractionation purifying?

Answer 21

Buffer and sucrose add density, ensuring nucleus forms pellet after centrifugation

Question 22

How is subcellular fractionation carried out by a centrifuge?

Answer 22

Using ultrasonication (sound waves to aggregate particles)
Mild detergent treatment
Differential centrifugation used to separate organelles

Question 23

What are the different centrifuge types?

Answer 23

• Refrigerated (Bench Top): separates whole cells at
  3000rpm
• Microcentrifuge: separates DNA and RNA at 20,000rpm
• Ultracentrifuge: subcellular fractionation & nucleur
  purification at 80,000rpm in vaccum
• High Speed centrifuge: Subcellular fractionation at
  20,000 rpm whilst refrigerated

Question 24

Which factors affect centifugal force?

Answer 24

Centrifugal force depends on

• Particle mass
• Acceleration

Question 25

What is a common use of isopycnic centrifuging?

Answer 25

isopycnic centrifugation in caesium chloride density gradients is another way of purifying and separating nucleic acids

Question 26

How are the different factors measured in a lab?

Answer 26

• Acceleration given as relative centrifugal force aka g units (xg)
• Spin meausred in rpm (revolutions per min)

Question 27

What is the function of the armoured bowl?

Answer 27

For safety and protection - prevent injury via rotor

Question 28

Why does a denser particle sediment faster?

Answer 28

The denser a particle is, the more compact it is

- therefore there is less drag and less buoyancy

Question 29

How are nucleic acids purified after centrifugation?

Answer 29

1. Add NaCl solution - DNA molecules repel eachother making it hard to separate
   - Na+ neutralise -ve charge on DNA
   backbone
2. Add ethanol to 70% final conc.
   - 70% is enough to ppt. DNA
   (if a higher % used other molecules
   will also ppt.)
3. Leave on ice for 5 mins
4. Centrifuge at 5- 10,000g (10,000 rpm for 3mins)
5. Remove supernatant
6. Rinse pellet in 70% ethanol and spin again
7. Air dry the pellet

Question 30

How can different types of DNA be differentiated using isopycnic banding?

Answer 30

• Circular supercoiled DNA (plasmids, mtDNA) bind
  weakly to EtBr compared to linear DNA
• EtBr also induces additional coiling
• Making plasmid DNA denser than linear DNA
  able to differentiate between the 2

Question 31

What is ethidium bromide?

Answer 31

EtBr is a fluorescent dye used to stain DNA aka isopycnic banding

Question 32

What does the term isopycnic mean?

Answer 32

When ultracentrifugal techniques make use of the difference in density between particles to separate them