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

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Presentation on theme: "Online Counseling Resource YCMOU ELearning Drive… School of Architecture, Science and Technology Yashwantrao Chavan Maharashtra Open University, Nashik."— Presentation transcript:

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

2 SEP-SBI084-CP01-03 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

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 minutes during Face-To-Face counseling.  Discussion about students difficulties or tutorial with assignments should follow the lecture for about 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 5 Learning Objectives After studying this module, you should be able to : Explain what is dialysis. State principle and technique of dialysis. State Factors affecting electrophoresis State nature of dialysis membrane Explain principle and technique of salting in Explain principle and technique ofsalting out

6 School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.6 Dialysis:1  Dialysis provides a means to change the buffer solution for a protein sample.  In dialysis, a protein solution is placed inside a bag consisting of a semi-permeable membrane.  The sample is then dialyzed against several changes of the desired final buffer.  First, the concentrated protein solution is placed in dialysis bag with small holes which allow water and salt to pass out of the bag while protein is retained.

7 School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.7 Dialysis:2  Next the dialysis bag is placed in a large volume of buffer and stirred for many hours (16 to 24 hours), which allows the solution inside the bag to equilibrate with the solution outside the bag with respect to salt concentration.  When this process of equilibration is repeated several times (replacing the external solution with low salt solution each time), the protein solution in the bag will reach a low salt concentration:

8 School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.8 Dialysis:3 This graphic illustrates the dialysis process.

9 School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.9 Dialysis:4 The graphic illustrates the complete dialysis process, except for it suggests you do this with distilled water.

10 School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.10 Dialysis:5  Buffer is used in this process to prevent the protein from denaturing due to the fact that distilled or deionized water is too low in salt  and may have an undesirable pH for your protein, which may cause it to denature.

11 School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.11 Dialysis of Purified Protein:1 1.Wear gloves any time you are doing a dialysis. 2.Get a large 2000ml beaker or larger if necessary 3.Estimate the volume of your sample and make a 100 fold excess amount of 10X buffer (If your sample volume is 10ml then make 1000ml of 10X buffer) 4.Place the dialysis buffer in the large beaker, cover with saran wrap, and place in the cold room until further use. 5.Get 2 dialysis clamps

12 School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.12 Dialysis of Purified Protein:2 6.Get enough dialysis bag to hold all of your sample. Each millimeter can hold about 1ml so measure out a length equal to the volume of your sample. 7.Get a new clean razor blade and make one clean cut at the end of the length you re going to use. 8.Rinse the orange clamps and the dialysis bag excessively with d H 2 O. Rinse the dialysis bag very well so that it becomes hydrated. 9.Once you have hydrated the dialysis bag you can roll your fingers along one of the ends until it opens.

13 School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.13 Dialysis of Purified Protein:3 10.Once you have one end of the dialysis bag open run lots of d H 2 O through it to rinse the inside and fully hydrate it. 11.Take one of the orange clamps and clamp one of the ends so that it is closed shut. 12.Fill the entire dialysis bag with water so that you can look for leaks. Apply a little pressure to the bag to look for small holes and to make sure that the clamp is working properly. 13.Grab the test tube samples from the cold room that you have decided to use according to your gel.

14 School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.14 Dialysis of Purified Protein:4 14.Use a powered pipette to collect all the fractions that you plan to dialyze and put the entire sample into the dialysis bag. 15.Take one of the orange clamps and clamp one of the ends so that it is closed shut. 16.Fill the entire dialysis bag with water so that you can look for leaks. Apply a little pressure to the bag to look for small holes and to make sure that the clamp is working properly. 17.Grab the test tube samples from the cold room that you have decided to use according to your gel.

15 School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.15 Dialysis of Purified Protein:5 18.Clamp the other side of the dialysis bad with the other orange clamp leaving a little room at the end so that the bag is easy to grab and work with. 19.Dialyze for three changes of buffer each going over 8 hours.

16 School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.16 Dialysis Membrane:1  The dialysis membrane is made of cellulose acetate and looks and feels like Saran wrap.  It is semi-permeable, meaning that molecules below a specified molecular weight can readily pass through the membrane whereas larger molecules cannot.  Dialysis membranes are available with a wide variety of molecular weight cutoffs; 10,000 and 40,000 dalton membranes are typically used for protein dialysis.  Once you have one end of the dialysis bag open run lots of d H 2 O through it to rinse the inside and fully hydrate it.

17 School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.17 Dialysis Membrane:2  Sometimes, proteins will precipitate during the dialysis process and may need to centrifuge the solution after dialysis to remove any particles which would interfere with the next step - such as ion-exchange chromatography where particles would clog the column and prevent the chromatography step from working.  In addition, you may lose enzyme activity during dialysis.

18 School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.18 Salting Out:1  This is the most common way to precipitate proteins.  Protein solubility is a complex function of the physiochemical nature of the proteins, pH temperature and the concentration of the salt used.  The process of salting out, the separation of an organic phase from an aqueous phase by the addition of a salt has been known for nearly a century.  This method is commonly used by biochemists in the purification of proteins.

19 School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.19 Salting Out:2  The most common type of precipitation for proteins is salt induced precipitation.  Protein solubility depends on several factors.  It is observed that at low concentration of the salt, solubility of the proteins usually increases slightly.  This is termed Salting in.  But at high concentrations of salt, the solubility of the proteins drops sharply.  This is termed Salting out and the proteins precipitate out.

20 School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.20 Salting Out:3  It also depends on whether the salt is Kosomtropic (stabilizes water structure) or Chaotropic (disrupts water structure).  At low concentrations of salt, solubility of the proteins usually increases slightly (salting in).  But at high concentrations of salt, the solubility of the proteins drop sharply (salting out).

21 School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.21 Salting Out:4  Initial salting in at low concentrations is explained by the Debye-Huckel theory. Proteins are surrounded by the salt counter ions (ions of opposite net charge) and this screening results in decreasing electrostatic free energy of the protein and increasing activity of the solvent, which in turn, leads to increasing solubility.  This theory predicts the logarithm of solubility to be proportional to the square root of the ionic strength.  The behavior of proteins in solutions at high salt concentrations was explained by Kirkwood.

22 School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.22 Salting Out:5  The abundance of the salt ions decreases the solvating power of the salt ions decreases the solubility of the proteins decreases and precipitation results.  At high salt concentrations, the solubility is given by the following empirical expression due to Cohn.  log S = B - KI  where S is the solubility of the protein, B is a constant (function of protein, pH and temperature) K is the salting out constant (function of pH, mixing and salt), and I is the ionic strength of the salt

23 School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.23 Salting Out:6  The log of protein solubility versus the salt concentration.  Different lines represent different proteins. Hence, by using the appropriate concentration range of the given salt, we can precipitate the protein of interest, preferentially form a mixture of proteins.

24 School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.24 Salting Out:7  The slope of the salting out curve is a function of the protein and salt involved, but it is not a function of the pH and temperature.  Also, as the molecular weight of the protein increases, the amount of salt required for precipitation decreases.  There is a series of the relative effectiveness of different anions in the salts used for protein precipitation.  This is referred to as the lyotropic of Hofmeister series.  The order is citrate > phosphate > sulphate > acetate which is about as good as chloride > nitrate > thiocyanate.  There is also a similar series for cations.

25 School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.25 What You Learn… You have learnt : Dialysis provides a means to change the buffer solution for a protein sample. Buffer is used in this process to prevent the protein from denaturing.  The dialysis membrane is made of cellulose acetate and it is semi-permeable in nature.  Salting out is the most common way to precipitate proteins.  At low concentrations of salt, solubility of the proteins usually increases slightly (salting in). But at high concentrations of salt, the solubility of the proteins drop sharply (salting out).

26 School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.26 Critical Thinking Questions 1.State why buffer is used in protein dialysis. 2.Explain the procedure of protein dialysis in details. 3.Give a detail account on salting in and salting out. 4.What is the role of dialysis membrane? © 2008, YCMOU. All Rights Reserved. 26

27 School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.27 Hints For Critical Thinking Question 1.To prevent the protein from denaturing. 2.After placing, a protein solution is placed inside a bag consisting of a semi-permeable membrane, how buffer is changed. 3.solubility of the proteins at low and high concentration. 4.Selective passage of molecules through the membrane. © 2007, YCMOU. All Rights Reserved.27

28 School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.28 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

29 School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.29 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

30 School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.30 Study Tips Microsoft Encarta Encyclopedia Wikipedia the free encyclopedia

31 End of the Presentation Thank You !


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