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Topic :- Cell FractionationSubmitted by :-

Mayank Kumar Gautam

Cell fractionationA host of fractionation procedures are employed by cell biologists. Each organelle has characteristics (size, shape and density for example) which make it different from other organelles within the same cell. If the cell is broken open in a gentle manner, each of its organelles can be subsequently isolated. The process of breaking open cells is homogenization and the subsequent isolation of organelles is fractionation. Isolating the organelles requires the use of physical chemistry techniques, and those techniques can range from the use of simple sieves, gravity sedimentation or differential precipitation, to ultracentrifugation of fluorescent labeled organelles in computer generated density gradient

Cell fractionation is required to study the biochemical nature and function of

various cell organelles such as chloroplast ,mitochondria,

endoplasmic reticulum ,nucleus ,ribosome, and macromolecules e.g.

proteins,carbohydrates,etc.It includes homogenization

and centrifugation

Homogenisation

• The cells may be broken by (a) grinding in mortar in homogenizer, or (b) exposing to ultrasonication at low temperature {4*C} in sugar or sucrose solution

• The cell wall and plasma membrane of the cell is broken carefully so that cell organelles may not be disrupted.

Homogenization is a process whereby a biological sample is brought to a state such that all fractions of the sample are equal in composition, i.e. a homogenized sample is mixed so well that removing some of the sample does not alter the overall molecular make-up of the sample remaining, and is identical to the fraction removed. Homogenization in biology is often followed by, or combined with, cell lysis and/or molecular extraction.

Centrifugation The homogenate contains various cell organelles. Their biochemical properties still retained. Different components of homogenate are separated on the basis of their shape, size, and density. The mixture of molecules are separated in a centrifuge. It has a specially designed rotor which is driven by a motor. It may be driven at definite speed r.p.m. the larged size organelles /molecules settled on bottom even at low rpm. The small sized molecule settled on bottom at high speed for the separation of cells components high speed centrifuge are used ;e.g., differential centrifuge, zonal centrifuge and density radiant centrifuge.

The preparative ultracentrifuge

Differential centrifuge

The organelles and macromolecules are of different shape, size and density. The large sized component will move faster to bottom as they experience the largest centrifugal force. At low speed the large component (nuclei, mitochondria)will sediment to form pellets at bottom of centrifuge tube. At higher speed ribosome and component of similar size are settled on the bottom of centrifuge tube. Further these component are separated by resuspending and separating the centrifugation process with gradually increasing speed.

Buoyant density gradient centrifugation

The cellular components independent of their size and shape are separated on the basis of their buoyant density. The sample is added with high concentrated of sucrose or cesium chloride (CsCl) at this point the component float and form a series of distinct bands in the centrifuge tube. The final band of equilibrium can be collected individually by puncturing the plastic tube and collecting drops from the bottom. This method is very sensitive and can separate macromolecules labeled with C-13 or N- 15 faster than labeled macromolecules

• It is used for separation of same size and different density. For example peroxisomes are separated from lysosome ,RNA from DNA , and agranular endoplasmic reticulum from granular endoplasmic reticulum•The terminal velocity is referred to as the sedimentation velocity of the particle and can be used to measure the size, weight or density of the particle.

Velocity sedimentation The mixture contains various sub-cellular components. In velocity sedimentation when homogenate is layered as a narrow band on top of a dilute salt solution that fills a centrifuge tube, the mixture is centrifuged. The sub-cellular components move as a series of distinct bands through salt solution each at different speed according to their size. They move in a process called velocity sedimentation. Then the centrifuge tube is filled with a shallow gradient of sucrose. It is prepared by a special mixing device.

This process prevents the convecting mixing of bands. The rate at which each component sediments is expressed in terms of sedimentation coefficient. The ultracentrifuge available in recent years rotates at the speed of about 80000 rpm and produces force above 50000 g. applying such forces small molecules can be allowed to sediment and separate from one another based on their size.

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