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**Hydrodynamic Techniques**

electrophoresis centrifugation Types of Centrifuges Ultracentrifuge -Analytical -Preparative High Speed Table Top Clinical Microfuges Fc = m2r Fb = -mo2r Ff = -fv

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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.

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**Analytical vs Preparative Centrifugation**

Sedimentation Coefficient (s) is the velocity per Fc, or s = v/2r units are the Svedberg (S), where 1 S = sec = m(p - s)/f Analytical vs Preparative Centrifugation

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**subcellular fractionation**

collect material cells precipitated macromolecules subcellular fractionation

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**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 = x 105 (rpm)2r [radius in cm]

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**Problems Density Gradients contamination resolution**

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®

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**Density Gradient Centrifugation**

Two Types: 1) Rate Zonal p > s separates primarily by mass 2) Isopycnic p < s equilibrium separates by density

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**Example of Isopycnic Separation**

Percoll, self-forming gradient light vs. heavy fraction

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**General Procedures 1) prepare gradient 2) apply sample 3) centrifuge**

step continuous self-forming (isopycnic) 2) apply sample layer on top bottom (isopycnic) dispersed (self forming) 3) centrifuge

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**4) collect and analyze fractions**

manually aspirate ‘bands’ fraction collector Measuring Density marker beads refractometer

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