1. Online Counseling Resource YCMOU ELearning Drive… School of Architecture, Science and Technology Yashwantrao Chavan Maharashtra Open University, Nashik – 422222, India

2. SEP-SBI084-CP01-02 Introduction Programmes and Courses  SEP-SBI084-CP01-U01

3. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.3 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 sonalisa_alkari@yahoo.co.in Sonalisaal@rediffmail.com

4. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.4 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.

5. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.5 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

6. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.6 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.

7. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.7 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.

8. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.8 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.

9. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.9 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

10. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.10 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.

11. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.11 Differential Centrifugation:3  The relative centrifugal force can be calculated by the following equation: R.C.F. = 1.119 x 10 -5 (rpm 2 ) 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).

12. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.12 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.

13. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.13 Differential Centrifugation:5

14. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.14 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.

15. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.15 Differential Centrifugation:7 Cell fractionation by differential centrifugation

16. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.16 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).

17. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.17 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.

18. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.18 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.

19. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.19 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.

20. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.20 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

21. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.21 What You Learn… You have learnt :  centrifugation is first step in a typical protein- purification 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

22. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.22 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. © 2007, YCMOU. All Rights Reserved.22

23. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.23 Hints For Critical Thinking Question 1.masses or densities 2.preparative technique, analytical technique 3.size, shape and density © 2007, YCMOU. All Rights Reserved.23

24. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.24 Study Tips:1  Book1 Title: Biophysical Chemistry (principles and techniques ) Author: Upadhay. Upadhay.Nath Publisher:Himalaya publishing House  Book2 Title: Physical Biochemistry (application to Biochemistry and molecular biology) Author: Freifelder Publisher: W. H. Freeman and Company

25. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.25 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

26. School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.26 Study Tips www.en.wikipedia.org Microsoft Encarta Encyclopedia http://en.wikipedia.org/wiki/ Wikipedia the free encyclopedia

27. End of the Presentation Thank You !