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

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Centrifuge rotors Fixed-angle axis of rotation At rest Swinging-bucket g Spinning g

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Geometry of rotors bc r max r av r min r max r av r min Sedimentation path length axis of rotation a r max r av r min

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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:

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

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

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Velocity of sedimentation of a particle v = velocity of sedimentation d = diameter of particle p = density of particle l = density of liquid = viscosity of liquid g = centrifugal force

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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.

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Size of major cell organelles Nucleus4-12 m Plasma membrane sheets3-20 m Golgi tubules1-2 m Mitochondria m Lysosomes/peroxisomes m Microsomal vesicles m

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Differential centrifugation of a tissue homogenate (I) 1000g/10 min Decant supernatant 3000g/10 min etc.

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

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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

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

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Differential centrifugation (V) Fixed-angle rotor: Shorter sedimentation path length g max > g min Swinging-bucket rotor: Long sedimentation path length g max >>> g min

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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.

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Density BarrierDiscontinuousContinuous Density gradient centrifugation

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How does a gradient separate different particles? Least dense Most dense

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

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

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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

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Buoyant density banding Equilibrium density banding Isopycnic banding

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Formats for separation of particles according to their density When density of particle < density of liquid V is -ve

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Discontinuous Resolution of density gradients ContinuousDensity Barrier III

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Problems with top loading

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

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