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Centrifugation Theory and

Practice

Routine centrifuge rotors

Calculation ofg-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

b c

rmax

rav

rmin

rmax

rav

rmin

Sedimentation path length

axis of rotation

a

rmax rav rmin

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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 lengththerefore have the lowest k-factors

The centrifugation times (t) and k-factors for two

different rotors (1 and 2) are related by:

2

211

k

tkt !

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Calculation ofRCFand Q

2

1000

18.11

QrxRCF

r

RCFQ 299!

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)

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

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g

d

v

lp

Q

VV

18

)(2

!

Velocity of sedimentation of a particle

v = velocityofsedimentation d= diameterofparticle

Vp = densityofparticle Vl= densityofliquid

Q = viscosityofliquidg = centrifugalforce

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Differential centrifugation Density of liquid is uniform

Density of liquid

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Size of major cell organelles Nucleus 4-12 Qm

Plasma membrane sheets 3-20 Qm

Golgi tubules 1-2 Qm

Mitochondria 0.4-2.5 Qm

Lysosomes/peroxisomes 0.4-0.8 Qm Microsomal vesicles 0.05-0.3Qm

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Differential centrifugation of a

tissue homogenate (I)

1000g/10 min

Decant

supernatant

3000g/10 minetc.

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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 ofpellets

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 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

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Differential centrifugation (V)

Fixed-angle rotor:

Shorter sedimentation path

length

gmax > gmin

Swinging-bucket rotor:

Long sedimentation path length

gmax >>> gmin

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Differential centrifugation (VI)

Rate of sedimentation can be modulated byparticle density

Nuclei have an unusually rapid

sedimentation rate because of their sizeAND high density

Golgi tubules do not sediment at 3000g, in

spite of their size: they have an unusuallylow sedimentation rate because of their verylow density: (Vp - Vl) becomes rate limiting.

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Density Barrier Discontinuous Continuous

Density gradient centrifugation

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How does a gradient separate

different particles?

Least dense

Most dense

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g

d

vlp

Q

VV

18

)(2 !

WhenVp > Vl :vis +veWhenVp = Vl :vis 0

Predictions from equation (I)

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gd

v lpQ

VV

18

)(2 !

WhenVp < Vl :vis -ve

Predictions from equation (II)

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Summary ofprevious slides

A particle will sediment through a

solution ifparticle density > solution

density

Ifparticle density < solution density,

particle will float through solution

When particle density = solution density

the particle stop sedimenting or floating

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Buoyant densitybanding

Equilibriumdensity banding

Isopycnic

banding

1

5

2

3

4

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1

2

3

3 Formats for separation ofparticles according

to their density

Whendensityofparticle < densityofliquidVis -ve

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Discontinuous

Resolution of density gradients

ContinuousDensity Barrier

I II

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Problems with top loading

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Vp >> Vl :vis +veforallparticles

throughoutthe

gradient

Separation ofparticles according to size