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

2. Introduction Programmes and Courses  SEP –SBI084– CP01-U01 SEP –SBI084– CP01-05 © 2008, YCMOU. All Rights Reserved

3. School of Science and Technology, Online Counseling Resource… Credits  Academic Inputs by Sonali Alkari Faculty YCMOU Nagpur Centre, Faculty LAD college P.G. D of Biotechnology Research officer Ankur Seeds Pvt Ltd sonalisa_alkari@yahoo.co.in Sonalisaal@rediffmail.com © 2008, YCMOU. All Rights Reserved

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: State principle and technique of electrophoresis State factors affecting electrophoresis Explain chemical ingredients and its roles Explain principle and technique of gel electrophoresis Explain principle and technique of isoelectric focussing

6. School of Science and Technology, Online Counseling Resource… Electrophoresis:1  Electrophoresis is the most known electrokinetic phenomenon.  It was discovered by Reuss in 1809.  He observed that clay particles dispersed in water migrate under influence of an applied electric field.  Generally, electrophoresis is the motion of dispersed particles relative to a fluid under the influence of an electric field that is space uniform.  Alternatively, similar motion in a space non-uniform electric field is called dielectrophoresis. © 2008, YCMOU. All Rights Reserved

7. School of Science and Technology, Online Counseling Resource… Electrophoresis: 2  Electrophoresis may be the main technique for molecular separation in today's cell biology laboratory.  Because it is such a powerful technique, and yet reasonably easy and inexpensive, it has become commonplace.  In spite of the many physical arrangements for the apparatus, and regardless of the medium through which molecules are allowed to migrate, all electrophoretic separations depend upon the charge distribution of the molecules being separated. © 2008, YCMOU. All Rights Reserved

8. School of Science and Technology, Online Counseling Resource… Electrophoresis:3  Electrophoresis can be one dimensional (i.e. one plane of separation) or two dimensional.  One dimensional electrophoresis is used for most routine protein and nucleic acid separations.  Two dimensional separation of proteins is used for finger printing, and when properly constructed can be extremely accurate in resolving all of the proteins present within a cell (greater than 1,500).

9. School of Science and Technology, Online Counseling Resource… Principles of Electrophoresis:1  Electrophoresis is the migration of charges particles or molecules in a medium under the influence of an applied electric field.  The usual purpose for carrying electrophoretic experiments are: i.To determine the number, amount and mobility of components in a given sample or to separate them. ii.To obtain information about electrical double layers surrounding the particles.  The modern day scientist use it for diverse purpose for determination of molecular weight of proteins to DNA sequencing.

10. School of Science and Technology, Online Counseling Resource… Principles of Electrophoresis:2

11. School of Science and Technology, Online Counseling Resource… Principles of Electrophoresis:3

12. School of Science and Technology, Online Counseling Resource… Factors Affecting Electrophoresis i.The sample: charge/mass ratio of the sample dictates its electrophoretic mobility. The mass consists of not only the size (molecular weight) but also the shape of the molecule. ii.Charge: the higher the charge, greater is the electrophoretic mobility. The charge however depends on pH of the medium. iii.Size: the bigger the molecule, greater are the frictional and electrostatic forces exerted upon by the medium of suspension. Consequently, large particle have a smaller electrophoretic mobility compared to the smaller particles. iv.Shape:rounded contours elicit lesser frictional and electrostatic retardation compared to shape contours.

13. School of Science and Technology, Online Counseling Resource… Techniques of Electrophoresis  There are two basic technique of Electrophoresis :  Free electrophoresis or electrophoresis without stabilizing media.  zone lectrophoresis or electrophoresis in stabilizing media.  Each of these techniques have their specific advantages, application and modes of operation.

14. School of Science and Technology, Online Counseling Resource… Gel Electrophoresis:1  Gel electrophoresis is a common laboratory technique that can be use both as preparative and analytical method.  Gel electrophoresisis the type of zone electrophoresis.  The principle of electrophoresis relies on the movement of a charged ion in an electric field.  In practice, the proteins are denatured in a solution containing a detergent (SDS).  In these conditions, the proteins are unfolded and coated with negatively charged detergent molecules. The proteins in SDS-PAGE are separated on the sole basis of their size.

15. School of Science and Technology, Online Counseling Resource… Gel Electrophoresis:2  Gel electrophoresis is a technique in which charged molecules, such as protein or DNA, are separated according to physical properties as they are forced through a gel by an electrical current.  Proteins are commonly separated using polyacrylamide gel electrophoresis (PAGE) to characterize individual proteins in a complex sample or to examine multiple proteins within a single sample.  There are two layers of gel, namely stacking or spacer gel, and resolving or separating gel.

16. School of Science and Technology, Online Counseling Resource… Gel Electrophoresis:3

17. School of Science and Technology, Online Counseling Resource… Modes of gel Electrophoresis Gel electrophoresis is usually carried out in any of the two modes i.Column electrophoresis ii.Slab gel electrophoresis

18. School of Science and Technology, Online Counseling Resource… Gel Electrophoresis:4  The term "gel" in this instance refers to the matrix used to contain, then separate the target molecules.  In most cases the gel is a crosslinked polymer whose composition and porosity is chosen based on the specific weight and composition of the target to be analyzed.  When separating proteins or small nucleic acids (DNA, RNA, or oligonucleotides) the gel is usually composed of different concentrations of acrylamide and a cross-linker, producing different sized mesh networks of polyacrylamide.

19. School of Science and Technology, Online Counseling Resource… Gel Electrophoresis:5  When separating larger nucleic acids (greater than a few hundred bases), the preferred matrix is purified agarose.  In both cases, the gel forms a solid, yet porous matrix. Acrylamide, in contrast to polyacrylamide, is a neurotoxin and must be handled using Good Laboratory Practices to avoid poisoning.  Proteins are usually analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE), by native gel electrophoresis, by quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE), or by 2-D electrophoresis.

20. School of Science and Technology, Online Counseling Resource… Procedure:1  The solution of proteins to be analyzed is first mixed with SDS, an anionic detergent which denatures secondary and non–disulfide–linked tertiary structures, and applies a negative charge to each protein in proportion to its mass.  Without SDS, different proteins with similar molecular weights would migrate differently due to differences in mass charge ratio, as each protein has an isoelectric point and molecular weight particular to its primary structure.  This is known as Native PAGE.  Adding SDS solves this problem, as it binds to and unfolds the protein, giving a near uniform negative charge along the length of the polypeptide

21. School of Science and Technology, Online Counseling Resource… Procedure:2  SDS binds in a ratio of approximately 1.4 g SDS per 1.0 g protein (although binding ratios can vary from 1.1-2.2 g SDS/g protein), giving an approximately uniform mass:charge ratio for most proteins, so that the distance of migration through the gel can be assumed to be directly related to only the size of the protein.  A tracking dye may be added to the protein solution to allow the experimenter to track the progress of the protein solution through the gel during the electrophoretic run.

22. School of Science and Technology, Online Counseling Resource… Separation:1  The denatured proteins are subsequently applied to one end of a layer of polyacrylamide gel submerged in a suitable buffer.  An electric current is applied across the gel, causing the negatively-charged proteins to migrate across the gel towards the anode.  Depending on their size, each protein will move differently through the gel matrix: short proteins will more easily fit through the pores in the gel, while larger ones will have more difficulty (they encounter more resistance).

23. School of Science and Technology, Online Counseling Resource… Separation:2  After a set amount of time (usually a few hours- though this depends on the voltage applied across the gel; higher voltages run faster but tend to produce somewhat poorer resolution), the proteins will have differentially migrated based on their size; smaller proteins will have traveled farther down the gel, while larger ones will have remained closer to the point of origin.  Thus proteins may be separated roughly according to size (and therefore, molecular weight)

24. School of Science and Technology, Online Counseling Resource… Chemical Ingredients and its Roles  '''Polyacrylamide gel (PAG)'' had been known as a potential embedding medium for sectioning tissues as early as 1954..  It possesses several electrophoretically desirable features that made it a versatile medium.  Polyacrylamide gel separates protein molecules according to both size and charge.  It is a synthetic gel, thermo-stable, transparent, strong, relatively chemically inert, can be prepared with a wide range of average pore sizes, can withstand high voltage gradients, feasible to various staining and destaining procedures and can be digested to extract separated fractions or dried for autoradiography and permanent recording.

25. School of Science and Technology, Online Counseling Resource… Sodium Dodecyl Sulfate  (SDS) (C12H25NaO4S; mw: 288.38).  SDS is the most common dissociating agent used to denature native proteins to individual polypeptides.  When a protein mixture is heated to 100 °C in presence of SDS, the detergent wraps around the polypeptide backbone.  It binds to polypeptides in a constant weight ratio of 1.4 g/g of polypeptide.  In this process, the intrinsic charges of polypeptides becomes negligible when compared to the negative charges contributed by SDS.

26. School of Science and Technology, Online Counseling Resource… Sodium Dodecyl Sulfate  Thus polypeptides after treatment becomes a rod like structure possessing a uniform charge density, that is same net negative charge per unit length.  Mobilities of these proteins will be a linear function of the logarithms of their molecular weights.

27. School of Science and Technology, Online Counseling Resource… Stacking Gel  The stacking gel is a large pore polyacrylamide gel (4%).  This gel is prepared with Tris buffer pH 6.8 of about 2 pH units lower than that of electrophoresis buffer.  These conditions provide an environment for Kohlrausch reactions, as a result, proteins are concentrated to several fold and a thin starting zone of the order of 19 μm is achieved in a few minutes.  This gel is cast over the resolving gel.  The height of the stacking gel region is always maintained more than double the height and the volume of the sample to be applied.

28. School of Science and Technology, Online Counseling Resource… Resolving Gel  The resolving gel is a small pore polyacrylamide gel (3 - 30%).  The Tris buffer used is of pH 8.8. In this gel, macro molecules separate according to their size.  Resolving gel were used for separating different range of proteins.  8% gel for 24 – 205 kD proteins,  10% gel for 14-205 kD proteins and  12% gel for 14-66 kD proteins

29. School of Science and Technology, Online Counseling Resource… Visualization:1  The results can be analyzed quantitatively by visualizing the gel with UV light and a gel imaging device.  The image is recorded with a computer operated camera, and the intensity of the band or spot of interest is measured and compared against standard or markers loaded on the same gel.  The measurement and analysis are mostly done with specialized software.  Depending on the type of analysis being performed, other techniques are often implemented in conjunction with the results of gel electrophoresis, providing a wide range of field-specific applications © 2008, YCMOU. All Rights Reserved

30. School of Science and Technology, Online Counseling Resource… Visualization:2  After the electrophoresis is complete, the molecules in the gel can be stained to make them visible.  Ethidium bromide, silver, or coomassie blue dye may be used for this process.  Other methods may also be used to visualize the separation of the mixture's components on the gel.  If the analyte molecules fluoresce under ultraviolet light, a photograph can be taken of the gel under ultraviolet lighting conditions.  If the molecules to be separated contain radioactivity added for visibility, an autoradiogram can be recorded of the gel. © 2008, YCMOU. All Rights Reserved

31. School of Science and Technology, Online Counseling Resource… Visualization:3 © 2008, YCMOU. All Rights Reserved

32. School of Science and Technology, Online Counseling Resource… Visualization:4  After staining, different proteins will appear as distinct bands within the gel.  It is common to run "marker proteins" of known molecular weight in a separate lane in the gel, in order to calibrate the gel and determine the weight of unknown proteins by comparing the distance traveled relative to the marker.  There are molecular weight size markers available that contain a mixture of molecules of known sizes.  If such a marker was run on one lane in the gel parallel to the unknown samples, the bands observed can be compared to those of the unknown in order to determine their size. © 2008, YCMOU. All Rights Reserved

33. School of Science and Technology, Online Counseling Resource… Buffer Systems:1  Many people continue to use a tris-glycine or "Laemmli" buffering system that stacks and resolves at a pH of ~8.3-9.0.  These pHs promote disulfide bond formation between cysteine residues in the proteins, especially when they are present at high concentrations because the pKa of cysteine ranges from 8-9 and because reducing agent present in the loading buffer doesn't co-migrate with the proteins.  Recent advances in buffering technology alleviate this problem by resolving the proteins at a pH well below the pKa of cysteine (e.g., bis-tris, pH 6.5) and include reducing agents (e.g. sodium bisulfite) that move into the gel ahead of the proteins to maintain a reducing environment. © 2008, YCMOU. All Rights Reserved

34. School of Science and Technology, Online Counseling Resource… Buffer Systems:2  An additional benefit of using buffers with lower pHs is that the acrylamide gel is more stable so the gels can be stored for long periods of time before use. © 2008, YCMOU. All Rights Reserved

35. School of Science and Technology, Online Counseling Resource… Two Dimensional Electrophoresis:1  Two-dimensional gel electrophoresis (2-D electrophoresis) is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissues, or other biological samples.  This technique separate proteins in two steps, according to two independent properties: the first- dimension is isoelectric focusing (IEF), which separates proteins according to their isoelectric points (pI);  the second-dimension is SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which separates proteins according to their molecular weights (MW). © 2008, YCMOU. All Rights Reserved

36. School of Science and Technology, Online Counseling Resource… Two Dimensional Electrophoresis :2  2DE is one of the best experimental tools for the reliable separation of thousands of proteins in a single gel.  2DE consists of a tandem pair of electrophoretic separations:  In the first dimension, proteins are resolved in according to their isoelectric points (pIs) using immobilized pH gradient electrophoresis (IPGE), isoelectric focusing (IEF), or non-equilibrium pH gradient electrophoresis (NEPHGE).  Under standard conditions of temperature and urea concentration, the observed focusing points of the great majority of proteins using IPGE (and to a lesser extent IEF) closely approximate the predicted isoelectric points calculated from the proteins' amino acid compositions © 2008, YCMOU. All Rights Reserved

37. School of Science and Technology, Online Counseling Resource… Two Dimensional Electrophoresis:3  This technique can provide molecular weight approximations (+/- 10%) for most proteins, with some dramatic exceptions.  Isoelectric focusing is the first step in two- dimensional gel electrophoresis, in which proteins are first separated by their pI and then further separated by molecular weight through SDS-PAGE.  In this way, complex mixtures consisted of thousands of different proteins can be resolved and the relative amount of each protein can be determined. © 2008, YCMOU. All Rights Reserved

38. School of Science and Technology, Online Counseling Resource… Two Dimensional Electrophoresis:4 © 2008, YCMOU. All Rights Reserved

39. School of Science and Technology, Online Counseling Resource… Isoelectric Focusing :1  Isoelectric focusing (IEF), also known as electrofocusing, is a technique for separating different molecules by their electric charge differences.  It is a type of zone electrophoresis, usually performed in a gel, that takes advantage of the fact that a molecule's charge changes with the pH of its surroundings.  A protein which is in a pH region below its pI will be positively charged and so will migrate towards the cathode.  However, as it migrates, the charge will decrease until the protein reaches a pH which is its pI. © 2008, YCMOU. All Rights Reserved

40. School of Science and Technology, Online Counseling Resource… Isoelectric Focusing:2  At this point it has no net charge and so migration ceases. Should it overshoot this point, it will enter a region of pH above its pI and so become negatively charged.  It will then reverse its direction of migration and now migrate towards the anode.  Therefore proteins become focused into sharp stationary bands with each protein positioned at a point in the pH gradient corresponding to its pI.  The technique is capable of extremely high resolution with proteins differing by a single charge being fractionated into separate bands. © 2008, YCMOU. All Rights Reserved

41. School of Science and Technology, Online Counseling Resource… What You Learn… You have learnt : Electrophoresis is the most known electrokinetic phenomenon Electrophoresis is the migration of charges particles or molecules in a medium under the influence of an applied electric field charge/mass ratio of the sample, Charge, shape and size are factors affecting electrophoresis. Slab gel and column electrophoresis are the different modes of electrophoresis. Free electrophoresis and zone electrophoresis are the basic techniques of electrophoresis. © 2008, YCMOU. All Rights Reserved

42. School of Science and Technology, Online Counseling Resource… What You Learn… You have learnt : Polyacrylamide gel separates protein molecules according to both size and charge SDS is the most common dissociating agent used to denature native proteins to individual polypeptides. Stacking gel is cast over the resolving gel. Isoelectric focusing is the first step in two- dimensional gel electrophoresis, in which proteins are first separated by their pI and then further separated by molecular weight through SDS-PAGE. Isoelectric focusing (IEF)is a technique for separating different molecules by their electric charge differences.

43. School of Science and Technology, Online Counseling Resource… Critical Thinking Questions 1.Describe in details the principle and technique of electrophoresis. 2.State how to analyze the results of electrophoresis. 3.What is isolectric focussing?. 4.Give detail account of two dimensional electrophoresis. © 2008, YCMOU. All Rights Reserved. 43

44. School of Science and Technology, Online Counseling Resource… Hints For Critical Thinking Question 1.migration of charges particles, factors affecting electrophoresis, Free electrophoresis and zone electrophoresis, Slab gel and column electrophoresis 2.Visualization, staining, marker proteins. 3.Separation of different molecules by their electric charge differences. 4.Isoelectric focusing, SDS-PAGE © 2007, YCMOU. All Rights Reserved.44

45. School of Science and Technology, Online Counseling Resource… 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

46. School of Science and Technology, Online Counseling Resource… 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

47. School of Science and Technology, Online Counseling Resource… Study Tips www.en.wikipedia.org Microsoft Encarta Encyclopedia http://en.wikipedia.org/wiki/ Wikipedia the free encyclopedia

48. School of Science and Technology, Online Counseling Resource… End of the Presentation Thank You !