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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