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PRINCIPLE
Rate of settling of a particle, or the rate of
separation of two immiscible liquids, is
increased many times by the application of
a centrifugal field (force) many times that
of gravity.
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FORCE IN A CENTRIFUGE IS
PROPORTIONAL TO TWO
THINGS
First, it depends on how fast the centrifuge
spins
Second, it depends on the radius of
rotation – think about “crack the whip”
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HOW FAST DOES A PARTICLE
SEDIMENT?
It depends on:
RCFs in the centrifuge
Size of particle
Particle density
Liquid density
Liquid viscosity
13. lseidman@matcmadison.edu
It turns out that if:
A particle is the same density as the liquid around
it, the particle doesn’t move
A particle is more dense than the liquid, it moves
down the tube
A particle is less dense than the liquid, it moves
up!
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On the next slide there is an excerpt from a
research article. Explain how centrifugation
is used in this research project.
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How Did Scientists Find Cytochrome C? Preparation of
Mitochondria from mouse liver
The mouse livers were removed after sacrifice and dounce homogenized in ice-cold
mitochondria isolation buffer (MIB) containing 250 mM mannitol, 0.5 mM EGTA, 5
mM HEPES, and 0.1% (w/v) BSA (pH 7.2) supplemented with the protease inhibitors
of leupeptin (1 mg/ml), pepstatin A (1 mg/ml), antipain (50 mg/ml), and PMSF (0.1
mM). Unbroken cells and nuclei were pelleted by centrifugation at 600g for 5 min at
4o
C. The supernatants were further centrifuged at 10,000g for 10 min at 4o
C to pellet
the mitochondria. The mitochondria pellet was resuspended in 4 ml MIB and loaded
onto a continuous Percoll gradient consisted of 30% (v/v) Percoll (Sigma), 225 mM
mannitol, 25 mM HEPES, 0.5 mM EGTA, and 0.1% (w/v) BSA (pH 7.2). The
suspension/gradient was centrifuged at 40,000g for 1 hr. The mitochondria were
removed from the brownish band at 1.10 g/ml with a transfer pipette. The
mitochondrial pellets were washed with MIB by centrifuging for 10 min at 6300g at
4o
C. The mitochondria were then resuspended gently in mitochondria resuspension
buffer containing 400mM mannitol, 10 mM KH2
PO4
, and 50 mM Tris-HCl (pH 7.2)
with 5 mg/ml BSA and stored on ice for up to 4 hr.
http://www.swmed.edu/home_pages/wanglab/Wanglab-pic/protocols.htm
19. lseidman@matcmadison.edu
INSTRUMENTATION
Lots of types
Some go faster and some slower
Some can take a lot of volume, others little
Some allow temperature control
Some allow you to add sample as centrifuge is
running
Some are specific for pathogens or whenever
aerosols must be avoided
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SAFETY!!
Centrifuges look sturdy, sort of like washing
machines
But, they are probably the most dangerous
instrument any of you will use
Also surprisingly easy to damage
BE CAREFUL!!!!!
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ROTORS ARE FRAGILE
Must withstand huge forces
In an ultracentrifuge, a 1 gram particle
“weighs” 0.65 tons
Any imperfection will weaken rotor
Therefore:
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PROPER USE OF ROTORS
Every rotor has a maximum speed
As high speed rotors age, derate
With ultrarotors retire them after certain age
or number of revolutions
Log books record every use and revolution
Overspeed discs on bottom
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Purchase the correct rotors for your
application
Derate them as necessary – follow
manufacturer’s directions
Balance, balance , balance
Check your textbook, p. 561 for guidelines
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BIGGEST CHALLENGES IS TO
READ THE CATALOGS
Rotor and tubes must match application
Rotor and tubes must match centrifuge
Rotor and tubes each have maximum RPMs
at which can use them – and may be different
Use the slower value
29. lseidman@matcmadison.edu
May need adaptors to fit certain tubes into
certain rotors
This is because rotors are engineered to take
varying size and styles of tubes
Makes them versatile, but also requires complex
combinations of adaptors and tubes
31. lseidman@matcmadison.edu
Follow manufacturer’s directions and NEVER
NEVER NEVER NEVER! spin faster than is
supposed to go
Select tubes that can handle the samples and
speeds you are using
Keep your hands and hair out of centrifuges!
Keep your hands out of rotors
Wear glasses
Make sure you know what you are doing
32. lseidman@matcmadison.edu
MUCH MORE SUBTLE, BUT
ALSO DANGEROUS,
Aerosols
Inevitable with normal centrifuge and very
high levels are released if accident occurs
Special centrifuges use special rotors, caps,
and seals that prevent leakage
Containment
