Transcript Centrifugation Theory and Practice

Centrifugation Theory and
Practice
• Routine centrifuge rotors
• Calculation of g-force
• Differential centrifugation
• Density gradient theory
Centrifuge rotors
axis of rotation
Swinging-bucket
Spinning g
At rest
g
Fixed-angle
Geometry of rotors
rmax rav rmin axis of rotation
a
rmin
rav
rmax
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
• 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:
k1t2
t1 
k2
Calculation of RCF and Q
 Q 2
RCF  11.18 x r 

 1000
RCF
Q  299
r
RCF = Relative Centrifugal Force (g-force)
Q = rpm; r = radius in cm
RCF in swinging-bucket and fixedangle rotors at 40,000 rpm
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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
Velocity of sedimentation of a particle
d (  p  l )
2
v
18
v = velocity of sedimentation
p = density of particle
g = centrifugal force
g
d = diameter of particle
l = density of liquid
 = viscosity of liquid
Differential centrifugation
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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
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Nucleus
Plasma membrane sheets
Golgi tubules
Mitochondria
Lysosomes/peroxisomes
Microsomal vesicles
4-12 m
3-20 m
1-2 m
0.4-2.5 m
0.4-0.8 m
0.05-0.3m
Differential centrifugation of a
tissue homogenate (I)
1000g/10 min
etc.
Decant
supernatant
3000g/10 min
Differential centrifugation of a
tissue homogenate (II)
1.
2.
3.
4.
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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
• 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)
• 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)
Swinging-bucket rotor:
Long sedimentation path length
gmax >>> gmin
Fixed-angle rotor:
Shorter sedimentation path
length
gmax > gmin
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 Barrier
Discontinuous
Continuous
How does a gradient separate
different particles?
Least dense
Most dense
Predictions from equation (I)
d (  p  l )
2
v
18
When p > l : v is +ve
When p = l : v is 0
g
Predictions from equation (II)
d (  p  l )
2
v
18
When p < l : v is -ve
g
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
1
Buoyant density
banding
2
3
4
5
Equilibrium
density banding
Isopycnic
banding
3 Formats for separation of particles according
to their density
1
2
3
When density of particle < density of liquid V is -ve
Resolution of density gradients
Density Barrier
I
II
Discontinuous
Continuous
Problems with top loading
Separation of particles according to size
p >> l : v is +ve
for all particles
throughout the
gradient