Centrifugation Flashcards
LOOK AT NOTES TO SEE HOW A CENTRIFUGE MACHINE LOOKS LIKE
LOOK AT NOTES TO SEE HOW A CENTRIFUGE MACHINE LOOKS LIKE
What are the two forms of rotors that can be found in a centrifuge?
- Switching out rotor: this takes large numbers of tubes which swing out so they are in line with the g-force i.e. goes horizontal.
- Fixed angle rotor: tubes go in at an angle but do not line at g-force- the pellet goes at the side of the tube instead of the bottom. Made of a solid piece of metal so can be spun at very higher speeds, much more than the switching out rotor.
What are the different types of centrifuges?
- (Refrigerated) Bench top centrifuge- Usually for separating whole cells and has typical speed up to 3000 rpm (revolutions per minute)
- Micro-centrifuge - typically for DNA and RNA, spins at 20,000 rpm
- High speed centrifuge (floor standing) – typically sub-cellular fractional with speeds up to 20,000 rpm – refrigerates as spinning heats it up
- Ultra-centrifuge – typically used for sub-cellular fractional and nucleic acid purification. Typical speeds up to 80,000 rpm. Rotor spins in a vacuum to avoid air resistance and frictional heating.
What does the rate of sedimentation depend on?
- Centrifugal force
- Buoyant density – if a particle is less dense than fluid it will float to the top. If it is denser it is going to go to the bottom of the tube but the frictional drag which will slow it down.
- Frictional drag
- The more massive (larger, heavier) the particle, the bigger the centrifugal force, so the faster it will sediment.
- For a given mass, the more dense the particle the faster it will sediment:
- More compact, so
- Less drag
- Less buoyancy
What does the centrifugal force depend on?
Force = mass x acceleration
What does acceleration depend on?
Acceleration = ω2r
So F = mass x ω2r
Ω/ ω = omega
What are the units for acceleration, force and mass?
- Acceleration is measured in m s-2
- Force is measured in Newtons
- Spin (angular velocity) Is measured in radians / se
- Acceleration is usually given as relative centrifugal force (RCF) also known as g units (x g)
- Spin is measured in revolutions per minute (rpm)
How do you convert RPM into angular velocity?
To convert rpm to angular velocity
- Divide by 60 to get revolutions per second
- Multiply by 2π to get radians per second
So ω = 2π x rpm / 60
acceleration = ω2r
= (2π x rpm / 60) 2 x r
= (4π2 x rpm2 / 3600) x r
Finally, to convert from Newtons to RCF, we divide by the force of gravity, 9.81 m s-1 or 981 cm s-1
* If we have measured r in cm, then we get
RCF = (4π2 x rpm2 x r) 3600 x 981
LOOK AT NOTES TO SEE HOW TO READ THE GRAPH
LOOK AT NOTES TO SEE HOW TO READ THE GRAPH
What are Svedburg units?
Historically one way to measure size of a macromolecule was to measure the sedimentation rate in an ‘analytical ultracentrifuge’. This rate was measured in Svedberg units.
Example
* Mammalian large ribosomal subunit 60S
* Mammalian small ribosomal subunit 40S
* But the whole Intact ribosome 80S not 100S
Note: Svedberg units are not S.I. units and are not additive
What might happen if cells are spun at too high a speed?
Mammalian cells are delicate. If centrifuged at high speed, cells may lyse due to shear forces. Typically they are centrifuged at 50-100g for 5-10 minutes.
How can mammalian cells be opened by?
Mammalian cells can be broken open by
* Mechanical stress in a homogeniser
* Ultrasonication
* Mild detergent treatment
Then use differential centrifugation to separate organelles
Microsomes: the ER breaks down into vesicles with a membrane
After centrifuge, how are the layers separated?
The nuclei have a higher density than other cell components. The bottom layer is buffer and sucrose to make it denser. It is centrifuged and the nucleus goes to the bottom forming a pellet. Others based on density can go as far as the interphase but not further as they are less dense than sucrose solution.
What are the two ways nucleic acid can be purified by
- purified from low MW contaminants and concentrated by ethanol precipitation
- isopycnic centrifugation in Caesium Chloride density gradients
How does purified from low MW contaminants and concentrated by ethanol precipitation work?
- Add NaCl solution.
a. Why? DNA molecules repel one another and so
difficult to separate. The Na+ ions neutralise
negative charged on sugar phosphate backbone
which has a negative charge. - Add ethanol to a final concentration of 70%
a. 70% is enough to precipitate DNA – if a higher
percentage is used then other molecules will
precipitate - Leave for 5 minutes on ice
- Centrifuge at 5-10,000g
a. E.g. 10,000rpm for 3 mins in a microfuge - Remove supernatant
- Rinse pellet in 70% ethanol and spin again
- Allow pellet to air dry
