SEPARATIONS: CENTRIFUGATION

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BASIC LABORATORY METHODS IN A REGULATED ENVIRONMENT

LECTURE OVERVIEW

• Basic theory
• Instrumentation
• Safety and proper operation

LECTURE OVERVIEW

• Basic theory
• Instrumentation
• Safety and proper operation

PRINCIPLE OF CENTRIFUGATION

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.

MANY APPLICATIONS

• Separate two immiscible liquids
• Isolate cellular organelles
• Isolate DNA, RNA, and proteins
• Isolate small particles including
• Bacteria
• Viruses
• Cells
• Industry: Separate cells that make a product from the product

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SUPERNATANT AND A PELLET

• Supernatant is the liquid at the top
• Pellet is particles at the bottom

Time

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” – person on the end of the line feels more force than person in center

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Painting by Winslow Homer of boys playing crack the whip

RELATIVE CENTRIFUGAL FORCE, RCF

• Also called x g; that is times gravity
• RCF = 11.17(r)(n/1000)²

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Where r = radius in cm from centerline

n = rotor speed in RPM, revolutions/minute

QUESTION, CALCULATING RCF

Suppose: r_min = 3.84 cm

r_average = 6.47 cm

r_max = 9.10 cm

n = 30,000 RPM

Then, what is the RCF

on a particle at

r_min, r_ave, and r_max?

ANSWERS

38,600 x g

65,043 x g

91,482 x g

• Don’t report RPM to other people; report RCF because everyone’s centrifuge is different

HOW FAST DOES A PARTICLE SEDIMENT?

• It depends on:
• RCFs in the centrifuge
• Size of particle
• Particle density
• Liquid density
• Liquid viscosity

HOW FAST DOES A PARTICLE SEDIMENT?

• It turns out that if:
• A particle is the same density as the liquid around it, the particle doesn’t move
• A particle is denser than the liquid, it moves down the tube
• A particle is less dense than the liquid, it moves up!

TWO BASIC MODES OF CENTRIFUGATION

• Most familiar is differential centrifugation
• There is also density gradient centrifugation

LECTURE OVERVIEW

• Basic theory
• Instrumentation
• Safety and proper operation

INSTRUMENTATION

• Lots of types of centrifuges
• 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

TERMINOLOGY

• Desktop, or clinical centrifuges <10,000 RPM
• Superspeeds, 10,000 – 30,000 RPMs (around 50,000 x g)
• Ultracentrifuges up to around 80,000 RPM and 500,000 x g

QUESTION

• On the next slide there is an excerpt from a research article. Explain how centrifugation is used in this research project.

How Did We 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 4^oC. 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 4^oC. 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.

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http://www.swmed.edu/home_pages/wanglab/Wanglab-pic/protocols.htm

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How Did We 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 4^oC. 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 4^oC. 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.

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http://www.swmed.edu/home_pages/wanglab/Wanglab-pic/protocols.htm

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LECTURE OVERVIEW

• Basic theory
• Instrumentation
• Safety and proper operation

SAFETY!!

• Centrifuges look sturdy, sort of like washing machines
• But they are perhaps the most dangerous instrument any of you will use
• Also, surprisingly easy to damage
• BE CAREFUL!!!!!

TWO MAIN SAFETY CONCERNS

• First is rotor coming off shaft – disaster
• If rotor leaves the centrifuge cabinet, it is like a bomb
• This type of accident can destroy a lab and harm anyone in the rotor’s path
• This is why the centrifuge housing of higher speed devices is extremely sturdy
• Second problem is leaking of hazardous materials during centrifugation, more subtle, also potentially dangerous

ROTORS ARE FRAGILE

• Must withstand huge forces
• In an ultracentrifuge, a 1 g particle “weighs” 0.65 tons
• Any imperfection will weaken rotor
• Therefore:

ROTORS ARE FRAGILE, REQUIRE

• 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 ultracentrifuge rotors retire them after certain age or number of revolutions
• Logbooks record every use and revolution
• Overspeed discs on bottom of rotors “tell” centrifuge the maximum speed for the rotor

PROPER USE OF ROTORS

• Purchase the correct rotors for your application
• Derate them as necessary – follow manufacturer’s directions
• Balance, balance, balance
• Tubes across from each other must match in weight
• For ultracentrifugation must also match in density
• Check your textbook for guidelines

PROPER USE OF ROTORS

• Protect rotors from:
• Scratches
• Moisture
• Spills
• Alkaline detergents (like Countoff, for radioisotopes)

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BIGGEST CHALLENGES IS OFTEN 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

READING CATALOGS

• 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
• Must make sure everything matches: centrifuge, rotor, tubes, adapters
• Use manufacturers’ catalogs to purchase all the pieces required
• Generally, purchase all accessories from the centrifuge manufacturer

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GENERAL RULES

• Follow manufacturer’s directions and NEVER NEVER NEVER NEVER! spin faster than system 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 safety glasses, even when operating relatively low speed centrifuges
• Make sure you know what you are doing

RELEASE OF AEROSOLS IS ALSO POTENTIALLY DANGEROUS

• Aerosols release
• Inevitable with normal centrifuge; very high levels are released if accident occurs
• There are centrifuges that use special rotors, caps, and seals that prevent leakage
• This is called “containment”

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