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Published byDanielle Fitzpatrick Modified over 2 years ago

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Hydrodynamic Techniques electrophoresis centrifugation Types of Centrifuges Ultracentrifuge -Analytical -Preparative High Speed Table Top Clinical Microfuges F c = m 2 r F b = -m o 2 r F f = -fv

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v = 2 rm ( p - s )/f The greater the centrifugal force ( 2 r), 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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Sedimentation Coefficient (s) is the velocity per F c, or s = v/ 2 r units are the Svedberg (S), where 1 S = sec = m ( p - s )/f Analytical vs Preparative Centrifugation

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collect material cells precipitated macromolecules subcellular fractionation

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Differential Centrifugation Relative Centrifugal Force expressed as x gravity RCF = F c /F g = 2 r/980 [ = (rpm)/30] RCF = x 10 5 (rpm) 2 r [radius in cm] prepare cell lysate subject to centrifugation centrifugal force time (g ·min) tube size and shape rotor angle separate supernatant and pellet re-centrifuge supernatant

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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 ®

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

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Measuring Density marker beads refractometer 4) collect and analyze fractions manually aspirate bands fraction collector

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