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
travel but initially experience lowest RCF • Smaller particles close to rmax have only a
short distance to travel and experience the highest RCF
离心机参数计算
Centrifuge rotors
axis of rotation Swinging-bucket
At rest g
Spinning g Fixed-angle
Geometry of rotors
rmax rav rmin axis of rotation a
rmin rav rmax
4-12 m 3-20 m 1-2 m 0.4-2.5 m 0.4-0.8 m 0.05-0.3m
Differential centrifugation of a tissue homogenate (I)
1000g/10 min
Decant supernatant
etc.
ቤተ መጻሕፍቲ ባይዱ
3000g/10 min
Rate of particle sedimentation depends mainly on its size and the applied g-force.
Size of major cell organelles
• Nucleus • Plasma membrane sheets • Golgi tubules • Mitochondria • Lysosomes/peroxisomes • Microsomal vesicles
rmin rav rmax
b
c
Sedimentation path length
k’-factor of rotors
• The k’-factor is a measure of the time taken for a particle to sediment through a sucrose gradient
l = density of liquid = viscosity of liquid
Differential centrifugation
• Density of liquid is uniform • Density of liquid << Density of particles • Viscosity of the liquid is low • Consequence:
Differential centrifugation (III) Expected content of pellets
• 1000g pellet: nuclei, plasma membrane sheets
• 3000g pellet: large mitochondria, Golgi tubules
• 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
t1
k 1t 2 k2
Calculation of RCF and Q
RCF11.18xr Q 2 1000
Q 299 RCF r
RCF = Relative Centrifugal Force (g-force) Q = rpm; r = radius in cm
RCF in swinging-bucket and fixedangle rotors at 40,000 rpm
• 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:
Velocity of sedimentation of a particle
v d2(p l ) g 18
v = velocity of sedimentation
p = density of particle
g = centrifugal force
d = diameter of particle
• 15,000g pellet: small mitochondria, lysosomes, peroxisomes
• 100,000g pellet: microsomes
Differential centrifugation (IV)
• Poor resolution and recovery because of: • Particle size heterogeneity • Particles starting out at rmin have furthest to