Fractionation of cells waseeem

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UNIVERSITY OF EDUCATION LAHORE
Fractionationof Cells

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Table of Contents
1 Fractionation of Cells............................................................................................................. 3
2 Principles of cell fractionation and ultracentrifugation as used to separate cell components......3
3 STEPS OF CELL FRACTIONATION............................................................................................. 4
3.1 EXTRACTION:................................................................................................................. 4
3.2 HOMOGENIZATION:.......................................................................................................4
3.2.1 Grinding:................................................................................................................ 4
3.2.2 High Pressure (FrenchPress or Nitrogen Bomb) and Osmotic shock:.......................... 5
3.2.3 Sonication(ultrasonic vibrations):............................................................................ 5
3.3 CENTRIFUGATION:.........................................................................................................5
4 The standard cell fractionation technique involvesfollowing methods: ....................................6
4.1 Differential velocity centrifugation(Velocity sedimentation) ............................................ 6
4.2 Equilibrium Density-gradient centrifugation(Equilibrium sedimentation):......................... 7
5 Centrifuges and Centrifuge Rotors:......................................................................................... 7
5.1 Micro centrifuges:..........................................................................................................7
5.2 High-speed centrifuges:..................................................................................................7
5.3 Ultracentrifuges:............................................................................................................ 8
5.4 Fixed angle rotors:.........................................................................................................8
5.6 Swinging bucket rotors:..................................................................................................8
6 APPLICATIONS:...................................................................................................................... 9
7 The Advantages & Disadvantages of Cell Fractionation............................................................ 9
7.1 Isolation:....................................................................................................................... 9
7.2 Reliability:..................................................................................................................... 9
7.3 Cell Death...................................................................................................................... 9
7.4 Time:............................................................................................................................. 9

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1 Fractionation of Cells
Although biochemical analysis requires disruption of the anatomy of the
cell fractionation technique has been devised to separate the various cell components while preserving
their individual functions. Just as a tissue can be separated into its living constituent cell types, so that
cell can be separated into its functioning organelles and macromolecules.
2 Principles of cell fractionation and ultracentrifugation as used to
separate cell components.
Cell fractionation is splitting cells up into its organelles.
 The tissue is chopped up and up into ice cold, isotonic, buffer solution.
 This is then put in a blender to break open the cells which is called 'homogenization'.
 The 'homogenate' is then filtered to get rid of debris like connective tissue.
 The mixture is spun on a centrifuge; the densest organelle will collect at the bottom.
 The separated bit at the bottom, the 'pellet' is left in the tube when the homogenate on top
which is called the supernatant is poured off into a new tube.
 This new tube is span again to collect the next densest organelle- this is repeated to collect the
desired organelles, with the speed increasing each time.
In step one the liquid is cold to slow down enzymes (that might have been freed from lysosomes) so
that they don't digest the organelles. It is isotonic to maintain normal water potential thereby
preventing organelles from bursting with water! Buffer solution maintains the PH so that it is
appropriate for the organelles.

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There are rules on how fast and long you have to spin the centrifuge to get the desired organelle
relating to the order of density. From most dense to least the order of these key organelles goes:
nucleus; mitochondria; lysosomes; ribosomes.
3 STEPS OF CELL FRACTIONATION
Cell fractionation involves 3 steps Extraction, Homogenization and Centrifugation.
3.1 EXTRACTION:
It is the first step toward isolating any sub-cellular structures. In order to maintain the biological
activity of organelles and bio-molecules, they must be extracted in mild conditions called cell-free
systems. For these, the cells or tissues are suspended in a solution of appropriate pH and salt content,
usually isotonic sucrose (0.25 mole/L) at0-40°C
3.2 HOMOGENIZATION:
The suspended cells are then disrupted by the process of homogenization.
It is usually done by:
3.2.1 Grinding:
Grinding is done by pester and mortar.

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3.2.2 High Pressure (French Press or Nitrogen Bomb) and Osmotic shock:
The later consists of two cylinders separated by a narrow gap.
3.2.3 Sonication (ultrasonic vibrations):
The shearing force produced by the movement of cylinders causes the rupture of cells. Ultrasonic
waves are produced by piezoelectric crystal. They are transmitted to a steel rod placed in the
suspension containing cells. Ultrasonic waves produce vibrations which rupture the cells. The liquid
containing suspension of cell organelles and ether constituents is called homogenate. Sugar or sucrose
solution preserves the cell organelles and prevents their clumping.
3.3 CENTRIFUGATION:
The separation (fractionation) of various components of the homogenate is carried out by a series of
cemrifugations in an instrument called preparative ultracentrifuge. The ultracentrifuge has a metal
rotor containing cylindrical holes to accommodate centrifuge tubes and a motor that spin the rotor at
high speed to generate centrifugal forces. Theodor Svedberg (1926) first developed die ultracentrifuge
which he used to estimate the molecular weight of hemoglobin.
Present day ultracentrifuge rotate at speeds up to 80,000 rpm (rpm= rotations per minute) and
generates a gravitational pull of about 500,000 g, so that even small molecules like t-RNA, enzymes
can sediment and separate from other components. The chamber of ultracentrifuge is kept in a high
vacuum to reduce friction, prevent heating and maintain the sample at 0-4°C.
During centrifugation, the rate at which each component settle down depends on its size and shape
and described in terms of sedimentation coefficient or Svedberg unit or S-value, where IS = 1 x 10-13
second.

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4 The standard cell fractionation technique involves following
methods:
4.1 Differential velocity centrifugation (Velocity sedimentation)
It is the first step of cell fractionation by which various sub-cellular organelles are separated based on
differences in their size. The homogenate in first filtered to remove unbroken cell clumps and
collected in a centrifuge tube. The filtered homogenate when centrifuged in a series of steps at
successively greater speeds, each step yields a pellet and a supernatant. The supernatant of each step
is removed to a fresh tube for centrifugation. For instance, at low speed (600g. for: 10 min) nuclear
fraction or pellet will sediment at medium speed (15,000g x 5 min) mitochondria fraction sediment
and at high speed (80,000 g. x 5 min.) micro-small fraction sediment. The final supernatant is soluble
fraction or cytosol.

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4.2 Equilibrium Density-gradient centrifugation (Equilibrium
sedimentation):
The organelle fractions (pallets) obtained in velocity centrifugation is purified by equilibrium density-
gradient centrifugation. In this method organelles are separated by their density not by their size.
The impure organelle fraction is layered on the top of a gradient solution, e.g., sucrose solution or
glycerol solution. The solution is more concentrated (dense) at the bottom of the centrifuge tube, and
decreases in concentration gradually towards the top. The tube when centrifuged at high speed the
various organelles migrate to an equilibrium position where their density is equal to the density of the
medium. Meselson, Stahl and Vinograd (1957) used denser cesium chloride gradient for separation of
a heavy DNA with 15N from DNA with 14N to provide evidence for semi-conservative DNA
replication.
In conclusion, we may say that what one can learn about cells, depends on the tools at one’s disposed
and, in fact, major advances in cell biology have frequently taken place with the introduction of new
too is and techniques to the study of cell. Thus, to gain different types of information regarding cell,
cell biologists have developed and employed various instruments and techniques. A basic knowledge
of some of these methods is earnestly required.
5 Centrifuges and Centrifuge Rotors:
There are three basic types of centrifuges used routinely by biologists. They differ in, among other
things, the rotational speed and relative centrifugal force that can be generated.
5.1 Micro centrifuges:
Micro centrifuge are table-top centrifuges used to process small volumes. They can attain speeds up to
approximately 12,000–13,000 rpm. They are typically used in cell culture, microbiology and
molecular biology.
5.2 High-speed centrifuges:
High speed centrifuges handle larger volumes and can attain higher speeds, up to approximately
30000 rpm. They come in both table-top and flow models.

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5.3 Ultracentrifuges:
Ultracentrifuge is designed to process moderate volumes of sample at speeds in excess of 70,000 rpm.
Ultracentrifuges are generally employed to isolate small particles, such as ribosomes and viruses and
macromolecules, such as proteins. They are also used in cell fractionation techniques that require
centrifugation of cellular components through relative high-density centrifugation media.
Centrifuge rotors are the highly-engineered devices that hold the centrifugation tubes as they are spun.
There are two basic types of rotors routinely used by biologists: fixed angle rotors and swinging
bucket rotors.
5.4 Fixed angle rotors:
Fixed angle rotors hold the centrifugation tubes at a fixed angle (generally 20 - 40 degrees) as they are
spun. These are the most commonly used rotors in the cell biology laboratory. In a fixed angle rotor,
the materials are forced against the side of the centrifuge tube, and then slide down the wall of the
tube, resulting in a faster separation of particles. They generally have no moving parts.
5.6 Swinging bucket rotors:
Swinging bucket rotors have buckets that are free to swing out on a pivot perpendicular to the axis of
rotation. They are they rotor of choice when using a density gradient centrifugation medium.
Moreover, if there is a danger or scraping off an outer shell of a particle (such as the outer membrane
of a chloroplast), then the swinging bucket is the rotor of choice. Swinging bucket rotors have hinges
that hold separate buckets, making this type of rotor more prone to mechanical failure.

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6 APPLICATIONS:
 It allows scientists to study functions and biochemical composition of cells and their
organelles
 Extraction of plasma membrane proteins and their functions.
 Membrane fractions are isolated from cell homogenate by density gradient centrifugation to
study their properties and functions
 Extraction of nuclear proteins and their functions
 Fractionation of sub-cellular proteins/ molecules.
7 The Advantages & Disadvantages of Cell Fractionation
Biologists often need to study certain organelles from a cell the mitochondria of human cell orthe chlo
roplasts of an algae or plant cell, for example
isolating these organelles involves a variety of procedures collectively called cell fractionatin. As a m
ethod for studying processes !ithinorganelles, cell fractionation has advantages and disadvantages.
7.1 Isolation:
With cell fractionation, biologists can isolate or purify specific organelles for furtherstudy. They can
carry out experiments with pure samples of these organelles that would beimpossible or more difficult
with the whole cell intact. Mitochondria, for example, could bepurified for use in experiments testing
how certain compounds affect the electron transportchain or oxidative phosphorylation (both of these
are part of the process that stores energyharvested from glucose in a form useful to the cell).
7.2 Reliability:
Reliable methods have been developed to isolate specific types of organelles from
cells. Typically a homogenate or mixture is prepared from a tissue sample; the homogenate can be cen
trifuged, spun in a test tube or centrifuge tube Ina machine with a whirling rotor that will throw the co
ntents of each tube outwards. Thisprocess separates the contents on the basis of their density.
Varying the speed of thecentrifuge or the length of time for which the contents are centrifuged, scienti
st’s canretrieve a sample of the organelles they want to study.
7.3 Cell Death
preparing a homogenate necessarily entails illing the cells. in many cases, this maynot be a disadvanta
ge% if a scientist is trying to study organelles within the cell, the death ofthe cell is immaterial.
On the other hand, once the cells are dead it's not possible to watchevents that would normally occur
in a live cell in real time. scientists often use other
techniques, like labeling with a fluorescent protein, to trace what happens in live cells.
7.4 Time:
Inmanyprocedures inbiological labs, cellfractionation issomewhattime+consuming. The samples m
ust be spunin the centrifuge forafairly lengthy periodof time toobtaingoodseparation% moreover
, they must often be spun several times, depending on theorganelle you are trying to isolate.
followingeachspin, the supernatant(the liquidabovethe sedimented debris or precipitate in the ce
ntrifuge tube) mustbe decanted withoutpouringoutthe precipitate, andthe precipitate mustbe re-
suspended if it contains thecomponent of interest.

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REFERENCE
 https://www.biologydiscussion.com/cell/cell-fractionation-extraction-homogenization-and-
centrifugation
 https://www.sciencedirect.com
 https://www.biologydiscussion.com