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principles and techniques of centrifugation (centrifugation (if there is…
principles and techniques of centrifugation
centrifugation
separate particles of macromolecules (cells, proteins, nucleic acids)
particle of higher density or larger in size travel at a faster rate
at the same time, the particle with less density and smaller in size float to the top and move at slow rate.
the greater the difference in density, the faster they move
if there is no difference in density:
isopyknic conditions
the particle stay ready
sedimentation
particles, including cells can be explain by the Stokes equation, which describes the movement of a sphere in a gravitational field
the rate of particle sedimentation is proportional to the particle size
the sedimentation rate is proportional to the difference in density between the particle and the medium
the sedimentation rate is zero when the particle density is the same as the medium density
the sedimentation rate decreases as the medium viscosity increases
the sedimentation rate increases as the gravitational force increases
formula for velocity of sedimentation :
centrifugation
involve spinning around an axis to produce a centrifugal force by increasing the magnitude of the gravitional force
the particle in the suspension experience a radial centrifugal force moving them away from the axis of rotation
centrifugal field :
angular velocity :
RCF
dependent on the speed of rotation in rpm and the distance of the particle form the center of rotation
speed - rpm (Q), distance cm - (r)
g force acting on particle is exponential to the speed of rotation
doubling the speed of rotation, increases the centrifugal force by a factor of four.
RCF formula :
types of centrifuge
differential centrifugation
simplest form of separation
type of separation is depend in the differences in the sedimentation rate of the biological particles of different size, shape and density
can be increased by using centrifugal force => yield a sireas of decreasing sedimentation rate
moving boundary centrifugation
repeat sedimentation at diff speed
medium - same density
the sedimentation speed is determined by the size, shape of the particle
application - low resolution separation such as penetration of nucleus
disadvantage - poor recoveries and contamination of particles
density gradient centrifugation
for purifying proteins and particularly nucleic acids
zonal centrifugation
-> sucrose density gradient centrifugation
isodensity centrifugation
-> caesium chloride density gradient centrifugation
rate-zonal centrifugation
preparation of gradient sucrose density for centrifugation medium (increase in density and velocity)
denser at the bottom; the less denser at the top
sample is applied in a thin zone at the top of the centrifuge tube on a density gradient
under centrifugal force, the particles will begin sedimenting through the gradient in separate zones according to their size and mass instead of density
insufficient time - incomplete separation; overtime - all particles will form pellet
sample - similar density, different molecular weight
isopycnic centrifugation
buoyant or equilibrium equilibrium
particles are separated solely on the density
the particle size only affect 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
gradient medium:
polyhydric alcohol (sugar)
polysaccharides
inorganic salts
iodinated compounds
colloidal silica
sample - similar molecular weight but different density (protein)
centrifuge rotors
fixed angle rotors
short distance to travel before pelleting
shorter run time
most widely used
swinging bucket rotor
longer distance to travel to allow better separation
easy to discard supernatant wo disturbing pellet
steps for extraction
to obtain pure organelles
the cells must be ruptured so the cells contents are released (cell membrane is broken but not the organelle
homogenization of the cell/ isolation of organelles
producing a liquified cellular homogenate
repeated centrifugation at progressively higher speed will fractionate homogenates of the cells into their components
the smaller the subcellular component, the greater is the centrifugational force required.
safety of using centrifugation
always ensure that load are evenly balanced before run
always observed the manufacturers mas speed and samples density rating
always observed speed reductions when running high density solutions, plastic adapters or stainless steel tubes