This document discusses cell fractionation, which is the process of separating cell components using centrifugation. It begins with an introduction to cells and their organelles. The history of cell fractionation is then discussed, noting Albert Claude's pioneering work in the 1930s. Methods for homogenizing and disrupting cells are described, including bead mills, ultrasonication, and enzymatic and osmotic lysis. Subcellular fractions are then isolated using differential and density gradient centrifugation based on properties like size, density, and sedimentation rate. Marker enzymes that are specific to certain organelles are used to identify fractions and assess purity. The document concludes with a summary of a typical cell fractionation protocol separating nuclei, mitochondria, microsome

2. HAIDER

3. CELL FRACTIONATION

4. Contents
 Introduction
 History
 Homogenization of tissues & cells
 Isolation of Subcellular fractions
 Marker Enzymes
 Summary

5. Introduction
 Cell is the structural and functional unit of life.
 Cell contain organelles which perform a variety of specific
functions.
 Electron micrographs explains only the structure but not
functions of the cell organelles.
 Toobtain precise information about the cell organelles, it is
necessary to isolate them free from contaminating organelles.

6. Contd
 Hence subcellular fractionation using centrifugation technique
is used.
 Individual organelle is identified using specific markers.
 This information is used to study potential cellular
irregularities and methods to correct them.

7. History
 Albert Claude in 1930 developed the
technique of cell fractionation &
identified the different organelles using
the technique of centrifugation. He
received Nobel Prize for the same in
1974.

8. Homogenization of tissues
& cells

9. Methods of homogenization
 Homogenizers fall broadly into two categories on the basis of
disruptive forces:
 Type 1 homogenizers: The material is subjected to the
disruptive force once.
 Eg: The French press is commonly used in the laboratory
for the homogenization of microorganisms particularly
those with a tough outer wall.

10.  Type 2 homogenizers: The material is repeatedly or
continuously exposed to the disruptive force.
1. Liquid Shear
2. Mechanical Shear
3. Sonication
4. Osmotic lysis
5. Other abrasive methods

11. Homogenization of cells
and tissues
 Homogenization just means to “breakopen”.
 Homogenization is concerned only with rupture of the
surface membrane.
 The principal aim is to achieve highest degree of cell
breakage using the minimum of disruptive forces without
damage to any of the organelles ofinterest.

12. Cell Disruption
Methods Cell disruption
methods
Mechanical
Solid Shear
Eg: Bead Mill
Liquid Shear
Eg:
Ultrasonication,
Non mechanical
Physical
Eg: Thermolysis,
Decompression,
Osmoticshock
Chemical
Eg: Antibiotics,
Detergents
Enzymatic
Eg: Lyticenzymes

13. Bead Mill
•It consists of a jacketed chamber with
a rotating shaft, running in its centre.
•Agitators are fitted with the shaft
which provide kinetic energy to the
small beads present in the chamber.
•Glass or ceramic beads often used to
crack open cells.
•The choice of bead size and weight is
greatly dependent on the type of cells.

14. •Disruption takes place due to the grinding action of the rolling beads and
the impact resulting from the cascading ones.
•Bead milling can generate enormous amounts of heat.
•Cryogenic bead milling : Liquid nitrogen or glycol cooled unit.
•Application: Yeast, animal and plant tissue.
•Small scale: Few kilograms of yeast cells per hour.
•Large scale: Hundreds of kilograms per hour.

15. Ultra Sonication
•Ultrasonic homogenizers work by inducing
vibration in a titanium probe that is
immersed in the cell suspension.
•A process called cavitation occurs, in which
tiny bubbles are formed and explode,
producing a local shockwave and disrupting
cell walls by pressure change.
•This method is very popular for plant and
fungal cells but comes at a disadvantage: It’s
very loud and has to be performed in an
extra room.
•Used in conjunction with chemical methods

16. Osmotic Shock

17.  By changing the solute concentration of the liquid surrounding
the cell.
 Through the process of osmosis, water can be moved into/out
the cell causing its volume to increase/decrease to the point
that results in cell bursts.
 Note that this method can only work with animal cells and
protozoa, since they do not have cell walls.

18. Detergents
•Directly damage the cell wall or
membrane, and this will lead to
release of intracellular content.
•Its mechanism of action is to
solubilize membrane proteins.
• Detergents can be anionic, cationic
and non-ionic detergents.
•Most commonly used anionic
detergent is sodium dodecyl sulfate
(SDS) which reorganizes the cell
membrane by disturbing protein-
protein interactions.

19. Enzymes
•Enzymes degrade the cell wall
components which will lead to
release of intracellular compounds.
•Enzymes that are commonly used
for degradation of cell wall of plants,
yeast and fungi include cellulases,
pectinases, xylanases and
chitinases.
•The enzyme’s high price and
limited availability limits their
utilization in large scale processes.

20. Isolation of Subcellular fractions

21.  The aim of subcellular fractionation is to separate organelles
with as little damage as possible.
 The methods of separation of cell organelles differ from tissue
to tissue.
 Approaches of subcellular fractionation:
1. Size: Difference in size result in differences in rate of
sedimentation.
 Problem: Individual mitochondria and microsomes derived from
different membrane systems are similar in size.
2. Surface Charge – Very limited use in practice
3. Density – Currently the most useful property for separation of
cell organelles.

22. Factors affecting organelle
density and size
 Density = Mass
Volume
 Neither mass nor volume of cell organelle are necessarily
constant.
 The easiest method for separating cell organelles according to
their density is to form a concentration gradient of some suitable
material, known as densitygradient solute.
 A particle suspended in a liquid of its own density neither floats
norsinkswhatever thecentrifugal field is applied.

23. Centrifugation
 The first analytical ultracentrifuge
was developed by Theodore
Swedberg in 1925.

24. Principle of centrifugation
 Particles suspended in a solution are pulled downward by
Earth’s gravitational force.
 The centrifuge works using the sedimentation principle,
where the centrifugal acceleration causes denser substances
and particles to move outward in the radial direction.
 In a solution , particles whose mass or density is higher than
that of the solvent sink or sediment and particles that are
lighter than it float on the top.
 Centrifugal force = mω2r
 ω is the angular velocity
 r is the distance from the centre of rotation

25. Centrifugal methods for the
separation of organelles
1. Separation by size – Differentialcentrifugation
2. Separation by density – Densitygradient
centrifugation

26. Differential Centrifugation
 Differential centrifugation separates particles basedon
difference in sedimentation rate, which reflect
differences in sizes anddensities.
 Steps:
1. Preparation of brokencells is poured in a centrifuge tube.
2. The preparation is initially centrifuged at low speeds to
completelysedimentthe largestand heaviestsub-cellular
component.
3. The supernatent is carefully decanted and is again
centrifuged ata higherspeeds till thedesired portion of cell
lysate isobtained.

28. Density Gradient Centrifugation
 Density gradient centrifugation is a variation of
differential centrifugation in which the sample is
centrifuged in a medium thatgradually increases in
density from top tobottom.
 Twotypesof densitygradient centrifugationare:
1. Rate Zonal Centrifugation
2. Isopycnic Centrifugation

29. Rate Zonal Centrifugation
 The particles are separated according to their size, shape, and
density or the sedimentation coefficient(s).
 Methods used for preparation of density gradients are
sucrose, glycerol, ficoll etc. 5-20% sucrose solution is
commonly used to form density gradient.
 The sample is applied in a thin zone at the top of the
centrifuge tube on a density gradient.
 If the density of the particle at any point in the gradient is
same to that of the gradient, then these particles will stop
otherwise it will move downward towards more denser region.
 Separation of particles depends on the duration of
centrifugation.***

31. Low Density
Medium Density
High Density

32. V. Marker
Enzymes

33.  An enzyme that is known to be localized exclusively in the
particular organelle.
 Examples-Acid phosphatase in lysosomes; Succinate
dehydrogenase in mitochondria.
 By monitoring where each enzyme activity is found during a
cell fractionation protocol, one can monitor the fractionation
of organelle protocol.
 Marker enzymes also provide information on the biochemical
purity of the fractionated organelles. The presence of
unwanted marker enzyme activity in the preparation indicates
the level of contamination by other organelles.

37. Summary
Homogenate
[in isotonicbuffer]
(Centrifuge at
2000*g for 10 min)
Pellet
[Nuclei]
(Marker Enzyme – DNA
Polymerase)
Supernatent
(Centrifuge at 10,000*g
for 20 min)
Pellet
[Mitochondria]
(Marker - Succinate
dehydrogenase and
Monoamineoxidase)
Supernatent
[Post Mitochondrial
Supernatent]
(Centrifuge at
1,05,000*g for 4hrs)
Pellet
[Microsome]
(Plasma membrane,ER,
Golgi, Lysosome,
Peroxisome)
Supernatent
[Cytosol containing
ribosomes and other
macromolecules]

38. THANKYOU