Transcript Online Counseling Resource YCMOU ELearning Drive…

Online Counseling Resource
YCMOU ELearning Drive…
School of Architecture, Science and Technology
Yashwantrao Chavan Maharashtra
Open University, Nashik – 422222, India
SEP-SBI084-CP01-02
Introduction
Programmes and Courses
 SEP-SBI084-CP01-U01
School of Science and Technology, Online Counseling Resource…
Credits
 Academic Inputs by
Sonali Alkari
 Counsellor, YCMOU Nagpur Study Centre,
 Faculty LAD college P.G. D of Biotechnology
 Research officer Ankur Seeds Pvt Ltd
 [email protected]
 [email protected]
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How to Use This Resource

Counselor at each study center should use this presentation to deliver
lecture of 40-60 minutes during Face-To-Face counseling.

Discussion about students difficulties or tutorial with assignments should
follow the lecture for about 40-60 minutes.

Handouts (with 6 slides on each A4 size page) of this presentation should
be provided to each student.

Each student should discuss on the discussion forum all the terms which
could not be understood. This will improve his writing skills and enhance
knowledge level about topics, which shall be immensely useful for end
exam.

Appear several times, for all the Self-Tests, available for this course.

Student can use handouts for last minutes preparation just before end
exam.
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Learning Objectives
After studying this module, you should be able to :
 Discuss principles of centifugation
 Describe differents types of centrifugation
 Discuss theory and principle Differential
centrifigation
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Introduction:Centrifugation:1
 The first step in a typical protein-purification scheme
is centrifugation.
 The principle behind centrifugation is that two particles
in suspension(cell, organelles or molecules) having
different masses or densities will settle to the bottom
of a tube at different rates.
 Proteins vary greatly in masses but not in density.
 Heavier or more dense molecule sediment more
quickly than lighter or les dense molecules; with time,
a pellet of molecules forms at the bottom of the tube.
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Introduction:Centrifugation:2
 The remaining liquid, the supernatant, contains
nonpelleted material.
 The average density of of protein is 1.37g/l. unless
a protein has an attached lipid or carbohydrates,its
density will not vary more than 15 percent from
volume.
 A centrifuge is an instrument that speds this
process by subjecting the particles to centrifugal
forces as great as 600,000 times the force gravity.
 The centrifugal force is roperational to the rotation
rate of the rotar(measured in revolution per minute
or rpm) and the distance of the tube from the
centre of the rotor.
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Centrifugation Uses
Centrifugation is used for two basic purpose:
1. As a preparative technique to separate one
type of material from other.
2. As as analytical technique to measure
physical properties (e.g., molecular weight,
density, shape and equilibrium binding
constants) of macromlecules.
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Differential Centrifugation:1
 This is the most common method of fractionating
cells
 Fractionation is the separation of the different
organelles within the cell .
 The organelles can be purified from a tissue
homogenate by differential centrifugation.
 This is a technique that is very commonly used in
cell biology to purify a specific target (i.e.,
organelle) from a lysate or homogenate of a whole
organism or tissue.
 The process of differential centrifugation is based
on the fact that organelles have differences in size,
shape and density and the gravity of a solution
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Differential Centrifugation:2
 As a result, the effect of gravity on each is
different.
 We can use this principle to separate an organelle
from a homogenous solution of particles by
artificially controlling.
 This is done by putting the solution in a variable
speed centrifuge and rotating them at a high rate
of speed.
 This creates a force that can be much greater than
the force of gravity, and particles that would
normally stay in solution will fall out and form a
pellet at the bottom of the tube.
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Differential Centrifugation:3
 The relative centrifugal force can be calculated by
the following equation:
R.C.F. = 1.119 x 10 -5 (rpm2) r
 where rpm is the revolutions per minute of the
rotor and r is the distance (in cm) of the particle
from the axis of rotation.
 The radius used is the distance from the center of
the axis of rotation to the middle of the centrifuge
tube.
 The forces created at low speeds are small (e.g.
600 X g) and only very large or dense particles will
fall out of solution (nuclei, whole cells and large
cellular debris).
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Differential Centrifugation:4
 At high speeds, the force created can be quite great
(e.g. as much as 300,000 X g).
 At these speeds, most particles will fall out of
solution and only very small, highly soluble
molecules will remain in solution.
 Differential centrifugation schemes involve stepwise
increases in the speed of centrifugation.
 At each step, more dense particles are separated
from less dense particles, and the successive speed
of centrifugation is increased until the target
particle is pelleted out.
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Differential Centrifugation:5
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Differential Centrifugation:6
 The final supernatant is removed, the pellet is
resuspended, and further study or purification can
be done on it.
 The fractionation of rat liver is an example of how
this process works:
 An important thing to note is that there is cross
contamination between the second and third
pellets.
 Mitochondria show up in Pellet 3 and lysosomes
show up in Pellet 2.
 This shows that the separations made by this
technique aren't absolute purifications, but relative
enrichments of organelles.
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Differential Centrifugation:7
Cell fractionation by
differential centrifugation
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Cell Fractionation by Differential Centrifugation:1
 Generally, the cellular homogenate is first filtered or
centrifuged at relatively low speeds to remove
unbroken cells.
 Then centrifugation of the homogenate at a slightly
faster speed or for a longer duration will selectively
pellet the nucleus — the largest organelle (usually 5
– 10 μm in diameter).
 A centrifugal force of 600 g (600 times the force of
gravity) is necessary to sediment nuclei; this is
generated by a typical centrifuge rotor operating at
500 revolutions per minute (rpm).
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Cell Fractionation by Differential Centrifugation:2
 The undeposited material (the supernatant) is next
centrifuged at a higher speed (15,000 g × 5 min),
which deposits the mitochondria, chloroplasts,
lysosomes,and peroxisomes.
 A subsequent centrifugation in the ultracentrifuge
(100,000 g × 60 min) results in deposition of the
plasma membrane, fragments of the endoplasmic
reticulum, and large polyribosomes.
 A force of 100,000 g requires about 50,000 rpm in
an ultracentrifuge; at this speed, the rotor chamber
is kept in a high vacuum to reduce heating due to
friction between air and the spinning rotor.
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Cell fractionation by Differential Centrifugation:3
 The recovery of ribosomal subunits, small
polyribosomes, and particles such as
complexes of enzymes requires additional
centrifugation at still higher speeds.
 Only the cytosol — the soluble aqueous
portion of the cytoplasm — remains
undeposited after centrifugation at 300,000
g for 2 hours.
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Method: 1
1. Cut tissue in an ice-cold isotonic buffer. It is
cold to stop enzyme reactions, isotonic to
stop osmosis and a buffer to stop pH
changes.
2. Grind tissue in a blender to break open
cells.
3. Filter to remove insoluble tissue.
4. Centrifuge filterate at low speeds (1000*g
for 10 min. This pellets the nuclei as this is
densest organelle.
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Method: 2
5. Centrifuge at medium speeds (10000* g
for 30 mins. This pellets mitochondria
whiach are the second densest organelle.
6. Centriguge at high speeds (100000*g for
30 mins. This pellets ER, golgi apparatus
and other membrane fragments.
7. Centriguge
at
very
high
speeds
(300000*g for 3 hrs. This
pellets
ribosomes
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What You Learn…
You have learnt :
 centrifugation is first step in a typical proteinpurification scheme.
 Different particales have masses or densities is the
principle of cenrifugation.
 Differential centrifugation is the most common
method of fractionating cells.
 The process of differential centrifugation is based
on the fact that organelles have differences in size,
shape and density and the gravity of a solution
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Critical Thinking Questions
1. State the principle of centrifugation.
2. Give a brief account on usages of
centrifugation.
3. Write a detail note on principles and
theory of differential centrifugation .
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Hints For Critical Thinking Question
1. masses or densities
2. preparative technique, analytical technique
3. size, shape and density
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Study Tips:1
 Book1
 Title: Biophysical
techniques )
Chemistry
(principles
and
 Author: Upadhay. Upadhay.Nath
 Publisher:Himalaya publishing House
 Book2
 Title: Physical Biochemistry (application
Biochemistry and molecular biology)
to
 Author: Freifelder
 Publisher: W. H. Freeman and Company
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Study Tips:2
 Book3
 Title: Essentials of Biophysics
 Author: Narayanan
 Publisher: New Age Int. Pub. New Delhi.
 Book4
 Title: A Text Book of Biophysics

Author: Roy R.N.
 Publisher:New Central Book Agency
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Study Tips
www.en.wikipedia.org
Microsoft Encarta Encyclopedia
http://en.wikipedia.org/wiki/
Wikipedia the free encyclopedia
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End of the Presentation
Thank You !