Cell fractionation is a technique used to isolate cell organelles. It involves homogenizing cells and separating the components via differential centrifugation or density gradient centrifugation. This allows organelles to be identified and studied individually using marker enzymes that are specific to each organelle. Cell fractionation has provided insights into the structure and function of organelles and their role in cellular processes.

1. Cell
Fractionation
& Marker
Enzymes
Dr. Praveen Katiyar

2. Cell

3. Cell Fractionation
• 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.
• Process of isolating cell organelles involves a variety of procedures. These
procedures are collectively called Cell fractionation.
• It can also be defined as : the separation of homogeneous sets , usually
organelles, from a larger population of cells.
• Individual organelle is identified using specific markers.
• This information is used to study potential cellular
irregularities and methods to correct them.

4. Cell Fractionation
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.
Methods of cell fractionation-
These involve -
1-Homogenization-The tissue is crushed between two rotating plates or
filtered through a
membrane or grinding with glass beads. Then the homogenate is run at low
speed to remove intact cells present in the supernatant.
2-Seperation of Cell organelles-
A-Differential centrifugation
B-Density equilibrium centrifugation

5. Cell Fractionation

6. Cell Fractionation
Steps
1. Homogenization
2. Differential centrifugation
3. Further separation and purification by density gradient
centrifugation
4. Collection of fractions
5. Analysis of fractions

7. Cell Fractionation
Homogenization
• First the cells must be broken open.
• A variety of different methods are available; the method chosen
depends on the type of experiment and the type of sample
(bacterial culture or mammalian tissue sample, for example)
Detergents like SDS or Triton X disrupt the cell membrane so the
contents can flow out.
• Subjecting the cells to ultrasound waves or sonicating them will also
break them open, as will agitation in the presence of metal or glass
beads.
• Blenders may work with tissue samples but will not work with
bacteria or other microorganisms.

8. Cell Fractionation
Homogenization or Cell disruption
Chemical : alkali, organic solvents, detergents
Enzymatic : lysozyme , chitinase
Physical : osmotic shock, freeze/thaw
Mechanical : sonication, homogenization, French press
Chemical Disruption-
Detergents such as TritionX-100 or NP40 can
permeabilize cells by solubilizing membranes.
Detergents can be expensive, denature proteins,
andmust be removed after disruption.

9. Cell Fractionation
Homogenization or Cell disruption
Sonication-
A sonicator can be immersed directly into
a cell suspension.
The sonicator is vibrated and high frequency
sound waves disrupt cells.
Homogenization-
Cells are disrupted as they pass
between the plunger and
vessel wall.

10. Differential centrifugation of homogenate
Homogenize cells in 0.25 M sucrose,
1 mM EDTA, 10 mM Tris-HCl, pH 7.4 1000g/10 min
pellet
nuclei
supernatant
supernatant
15,000g/15 min
100,000g/60 min
Pellet (LMF)
mitochondria
lysosomes
peroxisomes
pellet
vesicles (microsomes)
supernatant cytosol

11. Cell Fractionation
Differential Centrifugation
• Differential centrifugation is the process where a homogenate (soup
of tissue and cells)undergoes repeat centrifugations and increasing
centrifugal force.
• Centrifugations is the use of increased gravity to quicken the
precipitation of substances to the bottom.
• The tool used here is the centrifuge, "merry-go- round for test
tubes" that spin at various speeds.

12. Cell Fractionation
Differential Centrifugation
• The centrifuge separates the cell's parts into pellet and supernatant.
• The pellet are the large cell structures that are settled at the
test tube's bottom.
• The supernatant are smaller parts of the cell suspending in liquid,
the supernatant is decanted and undergoes another
centrifugation.
• The process is repeated and increases speed with each trial to
collect successively smaller parts of a cell in pellets.

13. Cell Fractionation
Differential Centrifugation

14. Cell Fractionation
Differential Centrifugation
• These are both test tubes
attached to a centrifuge.
The first picture is of a
test tube that has
undergone
homogenization but is
about to undergo
centrifugation .
• The second picture are
the results of the
centrifugation and
portrays the settling of
large cell parts.
01
02

15. Cell Fractionation
Buoyant Density Centrifugation
• The buoyant density centrifugation involves viruses with densities of
1.1-1.2 g/cm and a sucrose gradient.
• The cell suspension is added to the top of the sucrose gradient.
• In this centrifugation the densest componentsmove
fastest down the tube and stops at the sucrose density equal to
its own.
• The sucrose gradient bands at the bottom contain
cell components with high buoyant densities and the components
at the top have low buoyant densities.

16. Cell Fractionation
An illustration of the sucrose gradient and the buoyant
density centrifugation.

17. Cell Fractionation
Collection of fractions
Collecting Fractions-keeping samples pure and intact
1. By hand: puncture sidewall of centrifuge tube with needle
and withdraw fractions through syringe
2. Machine: gradient uploader; introduces very dense, non-miscible
medium into bottom of tube, pushes fractions up to be collected
from top
3. If no pellet, can collect fractions through hole in bottom of tube

18. Cell Fractionation
Analysis of fractions
Analysis of fractions-need to identify and quantify the purified
fractions, so that they can be used successfully in downstream
applications.
Methods:
1-Light or electron microscopy
2-Biochemical-determine presence of marker enzymes
3.Assay for a protein marker with an antibody (western)
4.Determine the protein concentration by using a spectrophotometer,
e.g. Bradfordassay
5.Determine specific activity (the ratio of activity of the enzyme
of interest to the protein concentration

19. Cell Fractionation
How is cell fractionation used in cell biology?
• Differential Centrifugation allows us to look at each
organelle within the cell.
• We can look at the individual organelles and study them in
detail.
• This helps to determine each organelles function within the
cell.

20. Cell Fractionation
Investigating Cell function
• Differential Centrifugation allows us to look at each
organelle within the cell
• We can look at the individual organelles and study them in
detail
• This helps to determine each organelles function within the
cell

21. Cellular Markers or Enzyme
Markers
• These are enzymes specific to certain organelles.
• Following the activity of these enzymes, it is possible to locate the
organelles.
• 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.
• However electron microscopy is the final step to determine purity of
the preparation and the presence of organelle in the sample.

22. Common Enzyme markers for
Organelles
Organelle Marker enzyme
Nucleus Lamin A & C
Mitochondria –Inner Membrane ATP synthetase
Mitochondria- Outer Membrane Monoamine oxidase
Mitochondria - Matrix Citrate synthase
Lysosome Cathepsin
Golgi bodies Galactosyl transferase
Microsome Glucose -6-phosphatase
Cytoplasm Lactate dehydrogenase
Peroxisomes Catalase