i am hafiz m waseem from vehari bsc in science college multan msc in university of education lahore pakistan

1. HAFIZ
MUHAM
MAD
WASEEM
lahore
Micrometry

2. Advanced Analytical Techniques

3. Introduction
 It is a process of rupturing the
cells and then separation into
the organelles to study their
structure, chemical
composition and functions
 Each organelle has
characteristics size, shape and
density which make it
different from other
organelles from the same cell
 This technique is devised to
separate various cell
components while preserving
their individual functions
 It allows to study different
parts of a cell in isolation

4.  It increases our knowledge of
organelle structure and
functions
 For example, it was
determined that a cell fraction
was rich in enzymes that
function in cellular respiration
 This unknown cell fraction was
found to be rich in
mitochondria and involved
with cellular respiration
 Although cell fractionation is
very gentle but it may result in
subtle changes to the cells
 It may involve enzymatic or
mechanical processes
Introduction

5. Advanced Analytical Techniques

6. Method of cell fractionation
 Different methods are used by
cell biologists for the cell
fractionation
 Starts with homogenization in
which cells are broken to
release their contents and is
followed by fractionating their
contents into different
components or cell organelles
 It involves the use of
techniques which range from
simple sieves, gravity
sedimentation or differential
precipitation to
ultracentrifugation for
separation of the fractions

7.  Main steps in cell fractionation
include
 Extraction. In this step the
cells or tissues are suspended
in suitable pH, salt
concentration and isotonic
medium
 Homogenization. It is
disruption of the cells or
tissues by grinding, sonication,
freeze/thawing, etc.
 Filtration. It is removal of
debris from the homogenate
 Centrifugation. In this step the
homogenate is separated due
to their densities
Method of cell fractionation

8. Advanced Analytical Techniques

9. Extraction
 It is first step in the way to cell
fractionation
 Here cell organelles and bio-
molecules are extracted in
mild conditions with minimum
damage to them
 Cells and organelles are
suspended at a suitable pH and
in isotonic medium
 Isotonic medium used is
generally sucrose at conc of
0.25 mol/L
 Different methods are used for
extraction of various
components or cell organelles
Phases of cell fractionation

10. Homogenization
 It is disruption of cells and
tissues in isotonic solution
which releases cellular
components which may contain
unbroken cells, pieces of cell
walls, cell membranes and
proteins such as enzymes
 Cell components are kept cold
and in isotonic form in a buffer
 Purpose is to prevent cell
organelles or bio-molecules
from osmotic damage,
degradation, etc.
 Protease or phosphatase
inhibitors are also used
Phases of cell fractionation

11.  Detergents used can interact
with both membranes and
parts of the cells that are
soluble in water
 Allow cellular components to
be mixed or homogenized
 It is combined with physical
methods like blenders, glass
beads or using sound energy
to lyse the cells
 It ensures that all of the cells in
the sample get broken and
isolation of cellular organelles
 Shear force must be carefully
controlled to avoid damage to
the cells/organelles
Phases of cell fractionation

12. Advanced Analytical Techniques

13. Filtration
 This step may not be
necessary and depends on
the source of cells under
investigation
 Animal tissue however is
likely to yield connective
tissue, debris, whole cells,
etc. which must be removed
 It is achieved either by
pouring through sieve with
or without some suction
pump
 Debris is discarded while the
clear filtrate is processed for
further analysis
Phases of cell fractionation

14. Centrifugation
 The homogenate is separated
in different fractions by
spinning it in a process called
centrifugation
 This imposes a centrifugal
force on the particles of the
homogenate perpendicular
to the axis of rotation
 As a result components of
the homogenate settle down
in the tubes on the basis of
their size and shape
 Each step yields a pellet and
supernatant which may be
centrifuged again
Phases of cell fractionation

15. Differential Centrifugation
 It is sequential centrifugation
of the homogenate at
increasing centrifugation force
 This results in isolation of
cellular components of
decreasing size and density
 This separation depends on
their sedimentation rate which
in turn is dependent on the
size and shape of the cellular
components
 The supernatant may be again
centrifuged to generate pellets
of lesser size and / or density
for further analysis
Phases of cell fractionation

16. Phases of cell fractionation

17. Advanced Analytical Techniques

18. Density gradient
centrifugation
 It is achieved by placing layer
after layer of gradient media in
a tube with the heaviest layer
at the bottom and the lightest
one at the top
 The homogenate is placed on
top of the layer and then
centrifuged at high speed
 The heaviest organelles move
down the tube fastest and stop
in the gradient medium with
density equal to their own
density
 This method is used to purify
sub-cellular organelles
Phases of cell fractionation

19.  Fractions can be recovered
by making a hole in the
bottom of the centrifuge
tube and then collecting a
series of the samples
 The tubes are numbered in
order from lighter to heavier
fractions/layers

20. Advanced Analytical Techniques

21.  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
Applications of cell fractionation