BIO 241
CELL BIOLOGY
LAB REPORT

NAME OF THE EXPERIMENT: Cellular Fractionation
EXPERIMENT NO:8
EXPERIMENT DATE:
SUBMITTED BY:
SUBMISSION DATE:
SUBMITTED TO: Elif Ikizler

Centrifugation first sediments those particles which are largest.In
addition,very asymmetrical molecules will sediment more slowly than spherical
particles of the same mass and density.Increasing either the centrifugaiton
speed or the time of centrifugation will cause smaller particles to pellet
also.Differential centrifugation separates particles not only according to the
size but also on the basis of density,since particles that are denser(eg.nuclei)will
pellet at a faster rate than less dense particles(eg.membranes)of the same
mass.Hence it is sometimes possible to obtain good separations of particles of
similar sizes but different densities by differential pelleting.The major problem
with differential pelleting is that,the centrifugal force necessary to pellet the
larger particles from the top of solution is also often sufficient to pellet the
smaller particles nearer the bottom of the tube.Hence in a single step it is only
possible to obtain a pure preparation of the smallest particles since these will
remain in solution after all the other larger particles have pelleted.The yield of
this procedure is likely to be low.This technique is usually used for the initial
processing of large volumes of heterogenious mixture to obtain fractions
enriched in the particles of interest prior to further purification.

In rate-zonal centrifugation,it is possible to avoid the problem of the co-
sedimentation of particles of different sizes by layering the sample as a narrow
zone onto the top of a density gradient.The primary purposes of the gradient
are to facilitate layering of the sample and to minimise convection currents in
the liquid column during centrifugation which would otherwise disrupt the
particle zones as they move down the tube.

Rate-zonal separations are ideal for the particles of defined
size(eg.proteins,RNA,ribozomes).However particles of the same type are
frequently heterogenious(eg.membrane fragments,mitochondria,and other cell
organelles).In this case,rate \u2013zonal separations do not efficiently separate the
particles according to type and it is more appropriate to separate particles on
the basis of some other parameter such as density.This can be done isopycnic
centrifugation.

In isopycnic separations,the particles are separated purely on the basis of
their density;size only affects the rate at which particles reach their isopycnic
positions.These separations are based on the centrifugation of particles in a
density gradient through which the particles move until their densities are the
same as that of the surrounding medium.The effective density of the particles
differs from one medium to another because particles are more hydrated in
some media than in others.

The features of isopycnic centrifugation are that because the separation
is an equilibrium one,prolonged centrifugation does not affect the separation as
long as the gradient remains stable and the particles are unaffected by
centrifugation.