Hydrodynamic Techniques• electrophoresis• centrifugationTypes of Centrifuges

• Ultracentrifuge -Analytical -Preparative• High Speed• Table Top• Clinical• Microfuges

Fc = m2rFb = -mo2rFf = -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• units are the Svedberg (S), where 1 S = 10-13 sec

= m(p - s)/f

Analytical vs Preparative Centrifugation

• collect material• cells• precipitated

macromolecules• subcellular

fractionation

Differential Centrifugation

Relative Centrifugal Force

• expressed as ‘x gravity’• RCF = Fc/Fg = 2r/980

[ = (rpm)/30]• RCF = 1.119 x 105 (rpm)2r

[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

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 CentrifugationTwo 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 Procedures1) prepare gradient

• step• continuous• self-forming

(isopycnic)

2) apply sample• layer on top• bottom (isopycnic)• dispersed (self

forming)

3) centrifuge

Measuring Density• marker beads• refractometer

4) collect and analyze fractions• manually aspirate ‘bands’• fraction collector

Subcellular Fractionationand Marker Enzymes

nuclei DNAmitochondria cytochrome oxidaselysosome hydrolasesperoxisome catalaseGolgi -mannosidaseplasma membrane adenylate cyclasecytosol lactate dehydrogenase