Methods of preparation of cell lysate and identification of subcellular components by marker enzymes

1. Pradeep Singh
M.Sc. 2nd Year
Department of Biochemistry
14 August,2018

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

3. I. 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.
 To obtain precise information about the cell organelles, it is
necessary to isolate them free from contaminating organelles.

4. 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.

5. II. 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.

6. III. Homogenization of tissues &
cells

7. 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.

8.  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

9. Homogenization of cells and
tissues
 Homogenization just means to “break open”.
 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 of interest.

10. Cell Disruption Methods
Cell disruption
methods
Mechanical
Solid Shear
Eg: Bead Mill
Liquid Shear
Eg:
Ultrasonication,
French Press
Non mechanical
Physical
Eg: Thermolysis,
Decompression,
Osmotic shock
Chemical
Eg: Antibiotics,
Detergents
Enzymatic
Eg: Lytic enzymes

11. 1. 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.

12. •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.

13. 2. 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

14. 3. French Press
•Primary mechanism: High shear
rates within the orifice
•Secondary mechanism:
Impingement
•Operating pressure: 10,000 to
50,000 psi
•Application: Small-scale recovery
of intracellular proteins and DNA
from bacterial and plant cells

15. 4. Thermolysis
 More common method in large scale release of proteins from
cells.
 Cells are exposed to high temperature shocks for short
durations immediately followed by longer exposure to lower
values in presence of buffer (1 mM MgCI2, 10 mM Tris-HCI pH
7.4)
 Periplasmic proteins in Gram Negative bacteria are released
when the cells are heated up to 50ºC.
 Cytoplasmic proteins can be released from E.coli within 10min
at 90 ºC.

16. 5. 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. 6. Chemical Solvents
 Often used with plant cells, organic solvents such as toluene, ether,
benzene, methanol, surfactants, and phenyl ethyl alcohol, DMSO (Dimethyl
sulphoxide) can be used to permeate cell walls.
 EDTA can be used specifically to disrupt the cell walls of gram negative
bacteria, whose cell walls contain lipopolysaccharides that are stabilized by
cations like Mg2+ and Ca2+. EDTA will chelate the cations leaving holes in
the cell walls.
 This method can be used with wide range of production organisms but the
problem can be that some proteins are denatured.

19. 7. 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.

20. 8. 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.

21. IV. Isolation of Subcellular fractions

22.  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.

23. 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 density gradient solute.
 A particle suspended in a liquid of its own density neither floats
nor sinks whatever the centrifugal field is applied.

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

25. 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

26. Centrifugal methods for the
separation of organelles
1. Separation by size – Differential centrifugation
2. Separation by density – Density gradient
centrifugation

27. Differential Centrifugation
 Differential centrifugation separates particles based on
difference in sedimentation rate, which reflect
differences in sizes and densities.
 Steps:
1. Preparation of broken cells is poured in a centrifuge tube.
2. The preparation is initially centrifuged at low speeds to
completely sediment the largest and heaviest sub-cellular
component.
3. The supernatent is carefully decanted and is again
centrifuged at a higher speeds till the desired portion of cell
lysate is obtained.

29. Density Gradient Centrifugation
 Density gradient centrifugation is a variation of
differential centrifugation in which the sample is
centrifuged in a medium that gradually increases in
density from top to bottom.
 Two types of density gradient centrifugation are:
1. Rate Zonal Centrifugation
2. Isopycnic Centrifugation

30. 1. 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.***

32. 2. Isopycnic Centrifugation
 Isopycnic centrifugation separates the particles solely on
the basis of buoyant density.
 Cesium chloride is used as a density gradient.
 This technique is used to separate particles of similar size
but different densities.
 It is also independent of time of centrifugation.
 Sedimentation of particles occur until the buoyant
density of particle is similar to the density of the
gradient.

33. Low Density
Medium Density
High Density

34. V. Marker Enzymes

35.  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.

38. Summary
Homogenate
[in isotonic buffer]
(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
Monoamine oxidase)
Supernatent
[Post Mitochondrial
Supernatent]
(Centrifuge at
1,05,000*g for 4 hrs)
Pellet
[Microsome]
(Plasma membrane, ER,
Golgi, Lysosome,
Peroxisome)
Supernatent
[Cytosol containing
ribosomes and other
macromolecules]

39. References
 Subcellular fractionation - A Cellular Approach by J.M. Graham
and D. Rickwood, Oxford University Press
 Wilson & Walker - Principles and Techniques of Biochemistry
and Molecular Biology, 7th Edition
 Stryer Biochemistry, 8th Edition
 Textbook of Biochemistry – DM Vasudevan, 8th Edition

40. THANK YOU