Hydrodynamic Techniques

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Hydrodynamic Techniques
electrophoresis centrifugation Types of Centrifuges Ultracentrifuge -Analytical -Preparative High Speed Table Top Clinical Microfuges Fc = m2r Fb = -mo2r 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.

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

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

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®

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

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