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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
AIM : To study techniques of homogenation and fractionation of cells
INTRODUCTION :
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Types of Centrifugal Separations:
Differential Pelleting:
Centrifugation first sediments those particles which are largest.Inaddition,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 thelarger 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.
Rate-zonal Centrifugation:
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.
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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 zonal separations do not efficiently separate the
particles according to type and it is more appropriate to separate particles onthe basis of some other parameter such as density.This can be done isopycnic
centrifugation.
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 isopycnicpositions.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.
Types of Centrifuge:
Low speed centrifuges;can be used for pelleting cells and faster
sedimenting cell organelles such as nuclei and chloroplasts.In addition,they
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can also be used for the fractionation of cells either on density gradients
or by centrifugal elutriation.
High speed centrifuges;used mainly for the preparation of subcellular
fractions.
Ultra centrifuges;used for quantitative estimations of sedimentationcoefficients of particles in sucrose gradients.
Miscelleneous centrifuges;used for larger volumes of sample.These
continuous-flow centrifuges can be used for pelleting,isopycnic
separations and phase separations.
Homogenisation:
a. Homogenisers: For most soft tissues such as rat liver,it is
common to use a liquid-shear technique to achieve cell
disruption.All liquid shear homogenisers involve the extrusion ofthe finely chopped tissue suspension(or cell suspension)through
the gap between a moving pestle and the wall of a glass outer
vessel.
b. Homogenisation Media: For the isolation of many organelles
(mitochondria,lysosomes,peroxisomes),it is important to maintain
an iso-osmotic medium to prevent damage due to osmotic
stress.The most commonly used homogenisation medium is 0.25M
sucrose buffered with Tris,Hepes or Tes ,to pH 7.4-8.0.However,
in certain cases,particularly for the isolation of the mitochondria,mannitol or sorbitol,either alone or in combination with sucrose,
are often used as the osmotic balancers.Gnerally, ionic solutions
are avoided since they remove peripheral proteins from
membranes.
c. Homogenisation of rat liver: The intact liver is a highly
vascularised organ and contains a considerable amount of blood.In
some cases,particularly for the processing of the nuclear pellet,
it is advisable to remove the blood by perfusion prior to
homogenisation.
Differential Centrifugation:
The simplest method for the separation of a homogenate into different
fractions is differential centrifugation.This involves sequential centrifugation of
the homogenate at increasing speeds to obtain a series of pellets containing
material of decreasing sedimentation rate.The main advantages of differential
centrifugation are that it is a rapid and simple technique ;is not limited by the
volume of the homogenate and the subcellular organelles are not stressed
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osmotically by exposure to hypertonic gradient media.The main disadvantage is
that the behaviour of the particles depends solely upon their sedimentation rate
through a medium of a single density and for a number of reasons,this will lead
to the formation of heterogenious pellets and the distriution of the same type
of organelle between different pellets.In density gradient centrifugation,a separation of the components of a
sample is achieved by sedimentation through a density gradient,that is a sloution
which inceraes in density with inceasing distance down the centrifuge tube.Two
distinct approaches are possible using this technique;rate-zonal centrifugation
and isopycnic centrifugation.
In rate-zonal centrifugation the sample is loaded as a narrow layer on top of
the density gradient.During centrifugation the sample particles separate as a
series of bands or zones,each with its characteristic sedimentation
rate.Centrifugation is halted before the particles pellet and the separatedcomponents are collected by fractionation of the gradient.The rate at which the
particles sediment depends on their size,shape and density and the centrifugal
force,density and viscosty profile of the gradient.
Microsome: Membranous vesicles from the endomembrane system form a
heterogenious collection of similar sized vesicles referred to as microsomes.
MATERIALS :
Fresh dissected rat liver
0.25M sucrose Vortex
Centrifuge
Centrifuge tubes
Blender
PROCEDURE :
1. A rat is dissected and its liver is taken out.
2. The rat liver is chopped into small pieces.3. 0.25 M sucrose is added and homogenized in a blender.
4. The homogenate is centrifuged at 3500 K for 5 min.
5. The supernatant,which is whole homogenate is collected and
5ml.of this is saved.
6. The rest of the homogenate is centrifuged at 3900K for 10 min.
7. The pellet is washed one time in 20ml.0.25M sucrose.
8. The nuclear pellet is resuspended and the suspention is saved.
9. The supernatent of steps 6 and 7 are combined.
10. They are centrifuged at 7000K for 10 min.11. The pellet is washed one time in 20 ml.0.25M sucrose.
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12. The mitochondrial pellet is resuspended.
13. The supernatents of steps 10 and 11 are combined
14. They are centrifuged at 16000K for 20 min.
15. The pellet is washed one time in 20 ml.0.25M sucrose
16. The lysosomal pellet is resuspended.17. The supernatents of steps 14 and 15 are combined.
RESULTS:
The pellet contains sedimented particles and the supernatent contains
unsedimented ones.
Fraction 1: cell debris,cytoskeleton
Fraction 2: Nuclear pellet (nuclei)Fraction 3: Mitochondrial pellet (mitochondria)
Fraction 4: Lysosomal pellet (lysosomes)
DISCUSSION :
The dissected rat liver is kept in cold ice bath and also during the
homogenization, otherwise the protein structure would be broken.
The resuspended pellets, final supernatant and the whole homogenate is
put in 20 degrees not to have deficient organelles for the next lab. Centrifuge
is so sensitive that the tubes must be in equilibrium and the tubes be equal inweight. Otherwise the rotor may be broken down.
Sucrose is used in order to increase the water intake of cells and to
increase the viscosity.
Pellets are washed with 0.25 M sucrose in order to minimize the
contamination.
REFERENCES :
Cell and Molecular Biology
Bio 241 Cell Biology Lab. Manual
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