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Hydrodynamic Techniques Types of Centrifuges • Ultracentrifuge -Analytical -Preparative • High Speed • Table Top • Clinical • Microfuges • electrophoresis • centrifugation Fc = m2r Fb = -mo2r Ff = -fv v = 2rm(p - s)/f • The greater the centrifugal force (2r), the faster the particle sediments. • The more massive a particle, the faster it moves in a centrifugal field. • The denser a particle, the faster in moves in a centrifugal field. • The denser the solvent, the slower the particle will move in a centrifugal field. • The particle velocity is 0 when the solvent density is greater than the particle density. • The greater the frictional coefficient (factors such as solvent viscosity, particle shape, etc.), the slower the particle will move. Sedimentation Coefficient (s) • is the velocity per Fc, or • s = v/2r = m(p - s)/f • units are the Svedberg (S), where 1 S = 10-13 sec Analytical vs Preparative Centrifugation • collect material • cells • precipitated macromolecules • subcellular fractionation Differential Centrifugation • prepare cell lysate • subject to centrifugation • • • • centrifugal force time (g ·min) tube size and shape rotor angle • separate supernatant and pellet • re-centrifuge supernatant Relative Centrifugal Force • expressed as ‘x gravity’ • RCF = Fc/Fg = 2r/980 [ = (rpm)/30] • RCF = 1.119 x 105 (rpm)2r [radius in cm] Problems • contamination • large particles contaminated with smaller particles • resolution • particles of similar sizes not separated • vibrations and convection currents Density Gradients • centrifugation in a dense medium • increases stability • provides greater resolution • common media: sucrose, CsCl, Ficoll®, Hypaque®, Percoll® Density Gradient Centrifugation Two Types: 1) Rate Zonal p > s • separates primarily by mass 2) Isopycnic p < s • equilibrium • separates by density Example of Isopycnic Separation • Percoll, self-forming gradient • light vs. heavy fraction General Procedures 1) prepare gradient 2) apply sample 3) centrifuge • step • continuous • self-forming (isopycnic) • layer on top • bottom (isopycnic) • dispersed (self forming) 4) collect and analyze fractions • manually aspirate ‘bands’ • fraction collector Subcellular Fractionation and Marker Enzymes nuclei mitochondria lysosome peroxisome Golgi plasma membrane cytosol DNA cytochrome oxidase hydrolases catalase -mannosidase adenylate cyclase lactate dehydrogenase Measuring Density • marker beads • refractometer
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