training-1new.ppt

8/10/2019 training-1new.ppt

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Centrifugation Theory and

Practice

Routine centrifuge rotors Calculation of g-force

Differential centrifugation

Density gradient theory


2/25

Centrifuge rotors

Fixed-angle

axis of rotation

At rest

Swinging-bucket

g

Spinning g


3/25

Geometry of rotors

b c

rmax

rav

rmin

rmax

rav

rmin

Sedimentation path length

axis of rotation

a

rmax rav rmin


4/25

k-factor of rotors

The k-factor is a measure of the time taken for aparticle 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

21

1

k

tkt


5/25

Calculation of RCFand Q

2

1000

18.11

QrxRCF

r

RCFQ 299

RCF = Relative Centrifugal Force (g-force)

Q = rpm; r = radius in cm


6/25

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


7/25

gd

v lp

18

)(2

Velocity of sedimentation of a particle

v = velocity of sedimentation d = diameter of particle

p= density of particle l= density of l iquid

= viscosity of l iquidg = centr ifugal force


8/25

Differential centrifugation

Density of liquid is uniform

Density of liquid


9/25

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 0.05-0.3m


10/25

Differential centrifugation of a

tissue homogenate (I)

1000g/10 min

Decant

supernatant

3000g/10 minetc.


11/25

Differential centrifugation of a

tissue homogenate (II)1. Homogenate1000g for 10 min

2. Supernatant from 13000g for 10 min

3. Supernatant from 215,000g for 15 min

4. Supernatant from 3100,000g for 45 min

Pellet 1nuclear

Pellet 2heavy mitochondrial

Pellet 3light mitochondrial

Pellet 4microsomal


12/25

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


13/25

Differential centrifugation (IV)

Poor resolution and recovery because of:

Particle size heterogeneity

Particles starting out at rminhave furthest to

travel but initially experience lowestRCF

Smaller particles close to rmaxhave only a

short distance to travel and experience the

highestRCF


14/25

Differential centrifugation (V)

Fixed-angle rotor:

Shorter sedimentation path

length

gmax> gmin

Swinging-bucket rotor:

Long sedimentation path length

gmax>>> gmin


15/25

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, inspite of their size: they have an unusuallylow sedimentation rate because of their verylow density: (p- l) becomes rate limiting.


16/25

Density Barrier Discontinuous Continuous

Density gradient centrifugation


17/25

How does a gradient separate

different particles?

Least dense

Most dense


18/25

g

d

v lp

18

)(2

When p> l: v is +veWhen p= l: v is 0

Predictions from equation (I)


19/25

g

d

v lp

18

)(2

When p< l: v is -ve

Predictions from equation (II)


20/25

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


21/25

Buoyant densitybanding

Equilibriumdensity banding

Isopycnic

banding

1

5

2

3

4


22/25

1

2

3

3 Formats for separation of particles according

to their density

When density of particle < density of l iquid V is -ve


23/25

Discontinuous

Resolution of density gradients

ContinuousDensity Barrier

I II


24/25

Problems with top loading


25/25

p

>>l

: v is +ve

for al l particles

throughout the

gradient

Separation of particles according to size

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