OUTLINE • Basic theory • Applications • Instrumentation
SEPARATIONS • This begins new topic: separations • Prior to this, talked about measurements and solution making
BIOSEPARATIONS • Separating and purifying biological materials • Filtration and centrifugation • Chromatography and electrophoresis also common methods
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.
MANY APPLICATIONS • Separate two immiscible liquids • Isolate cellular organelles • Isolate DNA, RNA, and proteins • Isolate small particles including • Bacteria • Viruses • Cells
SUPERNATANT AND A PELLET • Supernatant is the liquid at the top • Pellet is particles at the bottom
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”
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
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?
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
HOW FAST DOES A PARTICLE SEDIMENT? • It depends on: • RCFs in the centrifuge • Size of particle • Particle density • Liquid density • Liquid viscosity
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!
TWO BASIC MODES OF CENTRIFUGATION • Most familiar is differential centrifugation • Also density gradient centrifugation
On the next slide there is an excerpt from a research article. Explain how centrifugation is used in this research project.
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
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
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
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!!!!!
TWO MAIN SAFETY CONCERNS • First is rotor coming off shaft - disaster
ROTORS ARE FRAGILE • Must withstand huge forces • In an ultracentrifuge, a 1 gram particle “weighs” 0.65 tons • Any imperfection will weaken rotor • Therefore:
Expert design • Proper use • Retire at correct time • Derate (run slower) when necessary
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
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
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
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
GENERAL RULES • Protect the rotors from: • Scratches • Moisture • Spills • Alkaline detergents (like Countoff, for radioisotopes)
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
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