Lecture 3: Principles and technique of centrifugation

safety: mechanical stress

- always ensure that load are evenly balanced before run - always observe the manufacturers max speed and samples density ratings - always observe speed reduction when running high density solutions, plastic adapters, or stainless steel tube

steps for extraction to obtain pure organelles

- cells must ruptures (cell membrane broken) so the cell contents are released - process of rupturing cell is known as homogenization and the subsequent isolation of organelles is fractionation - ruptured cells producing a liquified cellular homogenate (enzymes, and membrane-bounded organelle) - repeated centrifugation at progressively higher speeds will fractionate homogenates of cells into their components - the smaller the subcellular component, the greater is the centrifugal force required to sediment it

density gradient centrifugation

- important technique for purifying proteins and particularly nucleic acids - two different types are zonal centrifugation and isodensity centrifugation

centrifuge rotor: swinging bucket rotor

- longer distance to travel (allow better separation) - easy to discard supernatant without disturbing the pellet

isopyknic (same density) centrifugation / buoyant / equilibrium separation

- particles are separated solely on the density - the particle size only affects the rate at which particles move until their density is the same as the surrounding gradient medium - the density of the gradient medium must be greater than the particles density (particles will never sediment to the bottom) - separation vary depending on the gradient medium used

zonal or rate-zonal centrifugation

- preparation of gradient sucrose density for centrifugation medium (increase in density and velocity) Den 1 < Den 2 < Den 3 - sample is applied in a thin zone at the top of the centrifuge tube on a density gradient (volume 10%) - under centrifugal force, particles will begin sedimenting through the gradient in separate zones according to their size and mass instead of density

particles in suspension experience

a radial centrifugal force that moves them away from the axis of rotation

overtime rate zonal centrifugation

all the particles will form pellet since the density of the particles is greater than the density of the gradient

sedimentation

can be explained by Stokes equation which describes the movement of a sphere in a gravitational field

RCF

dependent on the speed of rotation in rpm and the distance of the of the particles from the center of rotation

....... the speed of rotation increases the centrifugal force by a factor of four

doubling

The g force acting on particles is ............ to the speed of rotation

exponential

radial force

generated by the spinning rotor and expressed relative to the earth's gravitational force known as the relative centrifugal force (RCF) or g force

insufficient time rate zonal centrifugation

incomplete separation

centrifugal force ....... with the distance from the axis of rotation

increases

stokes equation

- rate of particle sedimentation is proportional to the particle size - sedimentation rate is proportional to the difference in density between the particle and the medium - sedimentation rate is zero when the particle density is the same as the medium density - sedimentation rate decreases as the medium viscosity increases - the sedimentation rate increases as the gravitational force increases

moving boundary centrifugation

- repeat sedimentation at different speed - medium same density (sedimentation speed depends mainly on size, shape of particles) - application: low resolution separation such as separation of nucleus, harvesting cells or producing crude subcellular fractions from tissue homogenate - disadvantages: poor recoveries and contamination of particles

centrifuge rotor: fixed angle rotor

- short distance to travel before pelleting - shorter run time - most widely used

differential centrifugation

- simplest form of separation (differential pelleting) - based on the difference in the sedimentation rate of the biological particles of different size, shape and density - can be increased by using centrifugal force (increasing of centrifugal force cycles will yield a series of decreasing sedimentation rate)

no density difference (isopyknic conditions)

particles stay steady

centrifuge

separate particles or macromolecules (cells, sub-cellular components, proteins, nucleic acid)

sample used in rate zonal centrifugation

similar density, different molecular weight (nucleic acid or cell organelle)

sample used in isopyknic scentrifugation

similar molecular weight but different density (protein)

centrifugation involve

spinning around an axis to produce a centrifugal force (increase the magnitude of the gravitational force)

dispersed system

subjected to artificially induced gravitational field

higher density particles or larger size

travel at a faster rate and at some point will be separated from particles less dense or smaller (float to top). The greater the density difference, the faster they move.