centrifugation. lseidman@matcmadison.edu outline basic theory applications instrumentation

CENTRIFUGATION

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OUTLINE

Basic theory Applications Instrumentation

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SEPARATIONS

This begins new topic: separations Prior to this, talked about measurements and

solution making

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BIOSEPARATIONS

Separating and purifying biological materials Filtration and centrifugation Chromatography and electrophoresis also

common methods

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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

Separate two immiscible liquids Isolate cellular organelles Isolate DNA, RNA, and proteins Isolate small particles including

Bacteria Viruses Cells

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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

Where r = radius in cm from centerline n = rotor speed in RPM, revolutions/minute

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CALCULATING RCF

Suppose rmin = 3.84 cm

raverage = 6.47 cm

rmax = 9.10 cm N = 30,000RPM Then, what is the 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 4oC 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

Desktop, or clinical centrifuges <10,000 RPM Superspeeds, 10,000 – 30,000 RPMs

(around 50,000 Xg) Ultracentrifuges up to 80,000 RPM and

500,000 Xg

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INSTRUMENT DESIGN

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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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TWO MAIN SAFETY CONCERNS First is rotor coming off shaft - disaster

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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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Expert design Proper use Retire at correct time Derate (run slower) when necessary

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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

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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

Protect the rotors from: Scratches Moisture Spills Alkaline detergents (like Countoff, for

radioisotopes)

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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