centrifugation

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Centrifugation Theory and Practice : 

Centrifugation Theory and Practice Routine centrifuge rotors Calculation of g-force Differential centrifugation Density gradient theory

Centrifuge rotors : 

Centrifuge rotors

Geometry of rotors : 

Geometry of rotors

k’-factor of rotors : 

k’-factor of rotors The k’-factor is a measure of the time taken for a particle to sediment through a sucrose gradient The most efficient rotors which operate at a high RCF and have a low sedimentation path length therefore have the lowest k’-factors The centrifugation times (t) and k’-factors for two different rotors (1 and 2) are related by:

Calculation of RCF and Q : 

Calculation of RCF and Q RCF = Relative Centrifugal Force (g-force) Q = rpm; r = radius in cm

RCF in swinging-bucket and fixed-angle rotors at 40,000 rpm : 

RCF in swinging-bucket and fixed-angle rotors at 40,000 rpm Beckman SW41 swinging-bucket (13 ml) gmin = 119,850g; gav = 196,770g; gmax = 273,690g Beckman 70.1Ti fixed-angle rotor (13 ml) gmin = 72,450g; gav = 109,120g; gmax = 146,680g

Slide 7: 

Velocity of sedimentation of a particle

Differential centrifugation : 

Differential centrifugation Density of liquid is uniform Density of liquid << Density of particles Viscosity of the liquid is low Consequence: Rate of particle sedimentation depends mainly on its size and the applied g-force.

Size of major cell organelles : 

Size of major cell organelles Nucleus 4-12 m Plasma membrane sheets 3-20 m Golgi tubules 1-2 m Mitochondria 0.4-2.5 m Lysosomes/peroxisomes 0.4-0.8 m Microsomal vesicles 0.05-0.3m

Differential centrifugation of a tissue homogenate (I) : 

Differential centrifugation of a tissue homogenate (I)

Differential centrifugation of a tissue homogenate (II) : 

Differential centrifugation of a tissue homogenate (II) Homogenate – 1000g for 10 min Supernatant from 1 – 3000g for 10 min Supernatant from 2 – 15,000g for 15 min Supernatant from 3 – 100,000g for 45 min Pellet 1 – nuclear Pellet 2 – “heavy” mitochondrial Pellet 3 – “light” mitochondrial Pellet 4 – microsomal

Differential centrifugation (III)Expected content of pellets : 

Differential centrifugation (III)Expected content of pellets 1000g pellet: nuclei, plasma membrane sheets 3000g pellet: large mitochondria, Golgi tubules 15,000g pellet: small mitochondria, lysosomes, peroxisomes 100,000g pellet: microsomes

Differential centrifugation (IV) : 

Differential centrifugation (IV) Poor resolution and recovery because of: Particle size heterogeneity Particles starting out at rmin have furthest to travel but initially experience lowest RCF Smaller particles close to rmax have only a short distance to travel and experience the highest RCF

Differential centrifugation (V) : 

Differential centrifugation (V)

Differential centrifugation (VI) : 

Differential centrifugation (VI) Rate of sedimentation can be modulated by particle density Nuclei have an unusually rapid sedimentation rate because of their size AND high density Golgi tubules do not sediment at 3000g, in spite of their size: they have an unusually low sedimentation rate because of their very low density: (p - l) becomes rate limiting.

Density gradient centrifugation : 

Density gradient centrifugation

How does a gradient separate different particles? : 

How does a gradient separate different particles?

Predictions from equation (I) : 

When p > l : v is +ve When p = l : v is 0 Predictions from equation (I)

Predictions from equation (II) : 

When p < l : v is -ve Predictions from equation (II)

Summary of previous slides : 

Summary of previous slides A particle will sediment through a solution if particle density > solution density If particle density < solution density, particle will float through solution When particle density = solution density the particle stop sedimenting or floating

Buoyant density banding Equilibrium density bandingIsopycnic banding : 

Buoyant density banding Equilibrium density bandingIsopycnic banding

3 Formats for separation of particles accordingto their density : 

3 Formats for separation of particles accordingto their density When density of particle < density of liquid V is -ve

Resolution of density gradients : 

Resolution of density gradients

Problems with top loading : 

Problems with top loading

Separation of particles according to size : 

p >> l : v is +ve for all particles throughout the gradient Separation of particles according to size

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