The document discusses advanced analytical techniques for cell fractionation, involving the rupture of cells to separate organelles for study. It details steps such as extraction, homogenization, filtration, and centrifugation, which are crucial for isolating cell components while minimizing damage. Techniques like differential and density gradient centrifugation are highlighted for their effectiveness in purifying sub-cellular organelles and analyzing their functions.

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