Centrifugation Flashcards
What are the different components of a centrifuge?
- refrigeration unit
- rotor
- motor
- armoured bowl
- armoured locking lid
What is the role of the rotor?
The rotor drives the centrifuge shaft
How can we convert N to relative centirfugal force?
Divide the force value by gravity (9.81ms⁻¹ )
can also use a nonogram
Which sized particle will sediment faster and why?
The bigger the particle the faster it will sediment as the centrifugal force will be bigger
What is the acceleration of a particle dependent on?
Acceleration is affected by
- distance from axis (r)
- spin rate squared (w²)
therefore acceleration = w² x r
so F = m x w²r
What does the rate of sedimentation depend on?
- centrifugal force
- buoyant density (less dense particles float)
- frictional drag
What is the Svedberg unit?
The measure of a particles size based on its sedimentation rate (how long it takes to form pellet)
not additive
How is rpm converted to angular velocity?
Divide by 60
Multiply by 2n
=> w = 2n x rpm/60
How is a centrifuge used to separate cells?
Mammalian cells are delicate can be separated using shear forces
at 50-100g
for 5-10 minutes
Outline the process of isopycnic centrifugation
- Centirufge at 100,000g for 16hrs
- RNA denser than DNA so forms pellet first
- DNA forms a band where CsCl density is equal to DNA
density
How is a centrifuge calibrated?
Balanced by eye
What is a microsome?
A fragment of ER and attached ribosomes obtained by centrifuging homogenised cells
What is subcellular fractionation?
Breaking open mammalian cells using mechanical stress in a homogeniser
(aka Ultracentrifugation)
What are the 2 forms of rotor in a centrifuge?
- Swing out rotor:
- Tubes swing out in line with G force (horizontally)
- Fixed angle rotor
- Tubes held at an angle, so pellet forms at side of tube
(not bottom)
- Tubes held at an angle, so pellet forms at side of tube
Which is the densest organelle in a cell?
The nucleus - forms pellet first
Why is caesium chloride used in isopycninc centrifugation?
Cs is a heavy metal
CsCl is a very soluble and dense copound
When spun in a centrifuge spontaneously forms a density gradient
Give an equation used to calculate the centrifugal force
Force = Mass x Acceleration
How can nuclear fractionation be further purified?
Via pellet ‘washing’
- Re-suspend pellet in buffer + sucrose
- Re centrifuge
- Density gradient centrifugation
What units are used in calculating the force?
Acceleration - ms⁻²
Force - N (newtons)
Spin - radiansˢ⁻¹ (angular velocity)
What is the role of the armoured lid?
Prevents the centrifuge opening whilst in use - preventing injury
What is the purpose of the buffer and sucrose in nuclear fractionation purifying?
Buffer and sucrose add density, ensuring nucleus forms pellet after centrifugation
How is subcellular fractionation carried out by a centrifuge?
Using ultrasonication (sound waves to aggregate particles)
Mild detergent treatment
Differential centrifugation used to separate organelles
What are the different centrifuge types?
- 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
Which factors affect centifugal force?
Centrifugal force depends on
- Particle mass
- Acceleration
