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OUTLINE
Basic theory
Applications
Instrumentation
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SEPARATIONS
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BIOSEPARATIONS
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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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MANY APPLICATIONS
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SUPERNATANT AND A
PELLET
Supernatant is the liquid at the top
Pellet is particles at the bottom
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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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RELATIVE CENTRIFUGAL
FORCE, RCF
Also = Xg
RCF = 11.17(r)(n/1000)2
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CALCULATING RCF
on a particle at
rmin, rave, and rmax?
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ANSWERS
38,600 X g
65,043 X g
91,482 X g
Don’t report RPM, report RCF because
everyone’s centrifuge is different
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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
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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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TWO BASIC MODES OF
CENTRIFUGATION
Most familiar is differential centrifugation
Also density gradient centrifugation
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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
4oC. The supernatants were further centrifuged at 10,000g for 10 min at 4 oC 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
4oC. The mitochondria were then resuspended gently in mitochondria resuspension
buffer containing 400mM mannitol, 10 mM KH2PO4, 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
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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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TERMINOLOGY
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INSTRUMENT DESIGN
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SAFETY!!
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TWO MAIN SAFETY
CONCERNS
First is rotor coming off shaft - disaster
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ROTORS ARE FRAGILE
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Expert design
Proper use
Retire at correct time
Derate (run slower) when necessary
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PROPER USE OF ROTORS
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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
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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
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GENERAL RULES
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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
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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
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