Centrifugation Handout

Sedimentation Theory A particle in a centrifugal field will experience 3 major forces: centrifugal force (Fc) = m2r 2 buoyant force (Fb) = -mo r frictional force (Ff) = -fv

where m = mass of the particle, = angular velocity, r = distance from the axis, mo = the mass of the displaced solution, f = frictional coefficient and v = velocity of the particle.

Centrifugal Force

The particle will move at a velocity such that the total force equals 0, therefore: m2r - mo2r - fv = 0 substituting ms = mo, where = partial specific volume of the particle (i.e., the reciprocal of the density of the particle or p) and s = density of the solvent, and solving for v results in: v = 2rm(1 - s)/f = 2rm(p - s)/f

This equation tells us several things about sedimentation: 1. 2. 3. 4. 5. 6. The greater the centrifugal force (2r) the faster the particle sediments. The more massive a particle (m), the faster it moves in a centrifugal field The denser a particle (p) the faster in moves in a centrifugal field. The denser the solution (s), the slower the particle will move in a centrifugal field. The greater the frictional coefficient (factors such as viscosity, particle shape, etc. influence this parameter), the slower the particle will move. The particle velocity is 0 when the solution density is greater than the particle density.

Analytical Centrifugation The velocity per unit force will be defined as the sedimentation coefficient (s), or: s = v/2r = m(p - s)/f When mass is expressed in g and f in g/sec s ranges from 10-13 to 10-11 sec. This is normally expressed in Svedberg (S) units where 1 S = 10-13 sec. Molecules and subcellular structures can be defined by a sedimentation coefficient which reflects there size, shape, and density.

Differential Centrifugation Preparative Centrifugation o takes advantage of the fact that more massive particles sediment faster than less massive particles o for example, organelles and other subcellular components can be isolated by differential centrifugation.

Differential Centrifugation prepare cell lysate subject lysate to centrifugation and separate supernatant and pellet fractions centrifugal force expressed as relative centrifugal force (RCF) or 'x g' (see appendix) product of time and g-force determines sedimentation tube size, shape and angle also affect sedimentation re-centrifuge supernatant at higher g-force problems contamination (larger particles contaminated with smaller particles) resolution (particles with similar properties difficult to separate) vibrations and convection currents

Density Gradient Centrifugation Centrifugation through a dense medium, or density gradient centrifugation, can increase the resolution and solve some problems associated with differential centrifugation. The two types of density gradient centrifugation are rate zonal and isopycnic (or equilibrium). rate zonal centrifugation: density of the particles being separated are greater than the density of the solvent. Separation is based upon mass (i.e., larger particles will sediment faster) isopycnic centrifugation: solvent density encompasses density of particles. The separation is based upon particle density.

General Procedures

1. Prepare Gradient discontinuous (step gradients), continuous (gradient maker), selfforming (isopycnic only) 2. Apply Sample layer on top (rate zonal or isopycnic) underlay on bottom (isopycnic only) dispersed in self-forming gradient media (isopycnic only) 3. Apply Centrifugal Force g-force and time depend on specifics of experiment centrifuge must be turned off before samples reach bottom of tube during rate-zonal centrifugation isopycnic centrifugation must achieve equilibrium 4. Collect and Analyze Fractions manually aspirate 'bands' or collect fractions if necessary determine density of fractions (refractomer or marker beads) specific enzymes or other biochemical properties can be used to evaluate particular subcellular compartments

Common Media Sucrose CsCl Ficoll Hypaque Percoll

Subcellular Fractionation and Marker Enzymes

nuclei mitochondria lysosome peroxisome Golgi plasma membrane cytosol DNA cytochrome oxidase hydrolases catalase -mannosidase adenylate cyclase lactate dehydrogenase

CENTRIFUGATION APPENDIX 1. RCF CALCULATION The relative centrifugal force (RCF) can be calculated from the following equation: RCF = (1.119 x 10-5)(rpm)2(r) where rpm is the speed of rotation expressed in revolutions per minute and r (radius) is the distance from the axis expressed in cm. The RCF units are "x g" where g represents the force of gravity. RCF can also be determined from the nomograph below. Place a straight edge to intersect the radius and the desired RCF to calculate the needed rpm. Alternatively place the straight edge on the radius and the rpm to calculate the g-force. For example, spinning a sample at 2500 rpm in a rotor with a 7.7 cm radius results in a RCF of 550 x g.