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Gel electrophoresis Techniques
The document is a comprehensive overview of electrophoresis, detailing its principles, mechanisms, and various methods including gel electrophoresis and polyacrylamide gel electrophoresis (PAGE). It discusses the role of factors like charge, size, and shape in protein separation, along with specific techniques such as isoelectric focusing and two-dimensional gel electrophoresis. Additionally, it covers the types of gels used, their applications, advantages, and disadvantages in the context of laboratory analysis of proteins and nucleic acids.
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Gel electrophoresis Techniques
- 1. GUIDED BY:- DR. ANILKUMAR ASSISTANT PROFESSOR DEPARTMENT OF LIFE SCIENCES, SCHOOL OF NATURAL SCIENCES, CENTRAL UNIVERSITY OF JHARKHAND Presented By:- Ashish Kumar Swain 19180402005 M.Sc. Life Sciences 2nd Semester Department of Life Sciences, School of Natural Sciences, Central University of Jharkhand 1
- 2. CONTENTS (as perSyllabus) Module 4: Electrophoresis: (a) General principle, factors affecting electrophoresis – voltage, current, resistance, buffer – composition, concentration, pH. (b) Gel electrophoresis; Types of gels (starch, agarose, polyacrylamide), Idea of electrophoresis Module, preparation of gel, sample application, running the samples, SDS-PAGE - Principle, apparatus and methods, gradient gels, Two dimensional gels isoelectric focusing. ASHISH KUMAR SWAIN 2
- 3. INTRODUCTION Electrophoresis isa process of migration of charged particle through a solution under the influence of external electric field. Electrophoresis of positively charged particles is sometimes called cataphoresis, while electrophoresis of negatively charged particles is sometimes called anaphoresis. Electrophoresis is used in laboratories to separate macromolecules based on size. Electrophoresis is used extensively in DNA, RNA and Protein analysis. When a potential difference applied between the two electrodes in a colloidal solution, it has been observed that the colloidal particles are carried to either the positive or the negative electrode. In other words, they behave as if they have electric charge present within them with respect to the dispersion medium. These phenomenon is known as Electrophoresis and may be defined as the migration of the colloidal particles through a solution under the influence of electric field. ASHISH KUMAR SWAIN 3
- 4. MECHANISM OF ELECTROPHORESIS ASHISHKUMAR SWAIN 4
- 5. PRINCIPLES OF ELECTROPHORESIS According to the laws of electrostatics, an ion with charge ‘Q’ in an electric field of strength ‘E’ will experience an electric force, 𝑭 𝒆𝒍𝒆𝒄𝒕𝒓𝒊𝒄𝒂𝒍 Hence, 𝑭 𝒆𝒍𝒆𝒄𝒕𝒓𝒊𝒄𝒂𝒍 = 𝐐. 𝐄 ……eqn-I The rate of resulting migration ‘V’ of the charged molecule through the solution is opposed by a frictional force 𝑭 𝑭𝒓𝒊𝒄𝒕𝒊𝒐𝒏𝒂𝒍 Hence, 𝑭 𝑭𝒓𝒊𝒄𝒕𝒊𝒐𝒏𝒂𝒍= V.f …….eqn-II Frictional Coefficient ‘F’ depends on Size & Shape of the migrating molecule & the viscosity of the medium. In constant electric field, combining these equation-I & II will be Q.E=V.f ASHISH KUMAR SWAIN 5
- 6. PRINCIPLES OF ELECTROPHORESIS The migration of the charged molecule in the electric field is generally expressed in terms of electrophoretic mobility (µ), which is the ratio of the migration rate of an ion to the applied electric field. 𝝁 = 𝝂 𝑬 = 𝑸 𝒇 As per the equation, if two molecules have the same mass & shape, the one with the greater net charge will move faster towards an electrode. FACTORS AFFECTING ELECTROPHORETIC MOBILITY:- Concentration of Agarose in Gel Shape & Size of Particles Conformation of Particles Charge of Particles Voltage current Time ASHISH KUMAR SWAIN 6
- 7. TYPES OF ELECTROPHORESIS ELECTROPHORESIS MOVINGBOUNDARY ELECROPHORESIS Capilary Electrophoresis Isotachophoresis Isoelectric Focusing Immunoelectrophoresis ZONE ELECTROPHORESIS Paper Electrophoresis Thin Layer Electrophoresis Gel Electrophoresis Cellulose Acetate Electrophoresis ASHISH KUMAR SWAIN 7
- 8. MOVING BOUNDARY ELECTROPHORESIS It is the electrophoresis in free solution. Hence, also known as Free Boundary Electrophoresis. It was developed by Tiselius in 1937. When an electric potential is applied across the tube, the charged molecules migrate towards one or the other electrodes. Because different charged molecules migrate at different rates, a number of interfaces or boundaries are formed between the leading edge of each charged molecules. ASHISH KUMAR SWAIN 8
- 9. ZONE ELECTROPHORESIS Itis an electrophoretic separation technique typically used for analysing protein, nucleic acids & biopolymers. During the process, different species in a sample are transported in a continuous electrolyte buffer system, subject to a potential gradient. Sample is constrained to move in a solid support like filter paper or a chemically inert gel matrix. This technique can used for both analytical & preparative purposes. ASHISH KUMAR SWAIN 9
- 10. GEL ELECTROPHORESIS Gelelectrophoresis is separation technique which uses the gel as a separating pocket. Molecules are separated in aqueous buffer supported within a polymeric gel matrix. Based on the molecular size of the substance molecular sieving technique is employed to facilitate the separation. Molecular sieving technique is the one in which electrophoretic mobility and migration of solute is purely depend on viscosity and pore size. Hence the molecular weight decide the migration of macromolecules in the system. ASHISH KUMAR SWAIN 10
- 11. TOOLS & PROCEDURESOF GEL ELECTROPHORESIS ASHISH KUMAR SWAIN 11
- 12. METHODS OF GELELECTROPHORESIS Gel Electrophoresis is carried out in two methods : 1. Horizontal gel electrophoresis. 2. Vertical gel electrophoresis ASHISH KUMAR SWAIN 12
- 13. HORIZONTAL GEL ELECTROPHORESIS In this technique the gel bed is placed in horizontal position as shown in fig. Both the ends of gel bed are connected with the electrophoresis buffer solution separately. The gel acts as the pocket in which the components with the smaller molecular size are trapped & it become easy to separate some of specific components. When potential difference is applied across the two ends, components of the mixture get separated on the basis of their electrophoretic ability. ASHISH KUMAR SWAIN 13
- 14. VERTICAL GEL ELECTROPHORESIS The technique employed here is also as similar as the Horizontal gel electrophoresis technique in case of principle, but the arrangement of the experiment is differing in these case. In these case, the sample is kept in the midpoint of the separation plate which is at 90 degrees with the ground. The separation is aided by the gravity and the efficiency of the separation is enhanced. ASHISH KUMAR SWAIN 14
- 15. TYPES OF GELELECTROPHORESIS GEL ELECTROPHORESIS AGAROSE GEL ELECTROPHORESIS STARCH GEL ELECTROPHORESIS POLYACRYLAMIDE GEL ELECTROPHORESIS ASHISH KUMAR SWAIN 15
- 16. TYPES OF GEL AGAROSEGEL Natural colloid extracted from sea weed. Linear polysaccharide made up basic repeat of unit Agarobiose. Gel casted horizontally Non-toxic. Separate large molecules (<20Kda) having large pore size Commonly used for DNA separations. Staining can be done before or pouring the gel. POLYACRYLMIDE GEL Cross-linked polymer of acrylamide & Methylenebisacrylamide. Polymerization initiated by Ammonium Persulfate & Catalysed by Gel casted vertically. Potent neuro-toxic. Separate small molecules. Used for DNA or protein separations. Staining can be done after pouring the gel. ASHISH KUMAR SWAIN 16
- 17. AGAR & AGAROSEGEL Agar is a mixture of poly saccharides extracted from sea weeds. Agarose is a highly purified uncharged polysaccharide derived from agar. Agarose is chemically basic disaccharide repeating units of 3,6-anhydro-L- galactose. Agarose dissolves when added to boiling liquid. It remains in a liquid state until the temperature is lowered to about 40° C at which point it gels. The pore size may be predetermined by adjusting the concentration of agarose in the gel. Agarose gels are fragile. They are actually hydrocolloids, and they are held together by the formation of weak hydrogen and hydrophobic bonds. The pores of an agarose gel are large, agarose is used to separate macromolecules such as nucleic acids, large proteins and protein complexes. ASHISH KUMAR SWAIN 17
- 18. STRUCTURE OF AGAR ASHISHKUMAR SWAIN 18
- 19. STRUCTURE OF AGAROSEGEL ASHISH KUMAR SWAIN 19
- 20. ADVANTAGES: Easy toprepare and small concentration of agar is required. Resolution is superior to that of filter paper. Large quantities of proteins can be separated and recovered. Adsorption of negatively charged protein molecule is negligible. It adsorbs proteins relatively less when compared to other medium. Sharp zones are obtained due to less adsorption. Recovery of protein is good, good method for preparative purpose. ASHISH KUMAR SWAIN 20
- 21. DISADVANTAGES & APPLICATION DISADVANTAGES Electro osmosis is high. Resolution is less compared to polyacrylamide gels. Different sources and batches of agar tend to give different results and purification is often necessary. APPLICATION Widely used in Immuno-electrophoresis. To separate different types of protein mixtures as well as nucleic acids. ASHISH KUMAR SWAIN 21
- 22. POLYACRYLAMIDE GEL ELECTROPHORESIS Itis prepared by polymerizing acryl amide monomers in the presence of methylene-bis-acrylamide to cross link the monomers. Structure of acrylamide (CH2=CH-CO-NH2) Polyacrylamide gel structure held together by covalent cross-links. Polyacrylamide gels are tougher than agarose gels. It is thermostable, transparent, strong and relatively chemically inert. Gels are uncharged and are prepared in a variety of pore sizes. Proteins are separated on the basis of charge to mass ratio and molecular size, a phenomenon called Molecular sieving. ASHISH KUMAR SWAIN 22
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- 24. PAGE PROCEDURES Thegel of different pore sizes is cast into a column inside a vertical tube, often with large pore gel at the top and small pore gel at the bottom. Microgram quantity of the sample is placed over the top of the gel column and covered by a buffer solution having such a pH so as to change sample components into anions. The foot of the gel column is made to dip in the same buffer in the bottom reservoir. Cathode and anode are kept above and below the column to impose an electric field through the column. Macromolecular anions move towards the anode down the gel column. There is no external solvent space, all the migratory particles have to pass through the gel pores. Rate of migration depends on the charge to mass ratio. Different sample components get separated into discrete migratory bands along the gel column on the basis of electrophoretic mobility and gel filtration effect. ASHISH KUMAR SWAIN 24
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- 26. VISUALIZATION OF GEL 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. ASHISH KUMAR SWAIN 26
- 27. TYPES OF PAGE PAGE SDS-PAGE NATIVE PAGE ASHISHKUMAR SWAIN 27
- 28. TYPES OF PAGE NATIVEPAGE Native gels are run in non-denaturing conditions, so that the analyte's natural structure is maintained. Separation is based upon charge, size, and shape of macromolecules. Useful for separation or purification of mixture of proteins. This was the original mode of electrophoresis. SDS-PAGE/DENATURED PAGE They are run in denaturing conditions. Separation is based upon the molecular weight of proteins. The common method for determining Molecular Weight of proteins. Very useful for checking purity of protein samples. ASHISH KUMAR SWAIN 28
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- 30. SDS-PAGE SDS-PAGE, sodiumdodecyl sulfate polyacrylamide gel electrophoresis, is a technique widely used in biochemistry, forensics, genetics and molecular biology to separate proteins according to their electrophoretic mobility. When a detergent SDS added to PAGE the combined procedure is termed as SDS PAGE. SDS coats protein molecules giving all proteins a constant charge to mass ratio. Due to masking of charges of proteins by the large negative charge on SDS binding with them, the proteins migrate along the gel in order of increasing sizes or molecular weights. SDS is an anionic detergent which denatures secondary and non–disulfide linked tertiary structures by wrapping around the polypeptide backbone. In so doing, SDS confers a net negative charge to the polypeptide in proportion to its length. Molecules in solution with SDS have a net negative charge within a wide pH range. A polypeptide chain binds amounts of SDS in proportion to its relative molecular mass. The negative charges on SDS destroy most of the complex structure of proteins, and are strongly attracted toward an anode in an electric field. ASHISH KUMAR SWAIN 30
- 31. SODIUM DODECYL SULFATE(SDS) ASHISH KUMAR SWAIN 31
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- 33. SDS-PAGE BEFORE SDS AFTERSDS ASHISH KUMAR SWAIN 33
- 34. PAGE APPARATUS SDS-coated largeproteins migrate slowly through the gel matrix and small proteins migrate quickly through the matrix The nearer the band to the well, the larger the molecular size ofprotein ASHISH KUMAR SWAIN 34
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- 36. APPLICATION OF SDS-PAGE Used for estimation of molecular weight of protein and nucleic acids. Determination of subunit structure of proteins. Purification of isolated proteins. Monitoring changes of protein content in body fluids Identifying disulfide bonds between protein Quantifying proteins Blotting applications ASHISH KUMAR SWAIN 36
- 37. STARCH GEL ELECTROPHORESIS A suspension of granular starch should be boiled in a buffer to give a clear colloidal suspension. The suspension on cooling sets as a semisolid gel due to intertwining of the branched chains of amylopectin. In order to avoid swelling and shrinking petroleum jelly is used. ADVANTAGES: High resolving power and sharp zones are obtained. The components resolved can be recovered in reasonable yield especially proteins. Can be used for analytical as well as preparative electrophoresis. DISADVANTAGES: Electro osmotic effect. Variation in pore size from batch to batch. ASHISH KUMAR SWAIN 37
- 38. ISOELECTRIC FOCUSING (IEF) It is an electrophoretic method in which proteins are separated on the basis of their Isoelectric Points (pIs). It makes use of the property of proteins that their net charges are determined by the pH of their local environments. If the pH gradient is equal to its pI, Its net charge will change. Focusing is a steady state mechanism with regard to pH . Proteins approach their respective pI values at differing rates, but remain relatively fixed at those pH values for extended periods. Proteins are positively charged in solutions at pH values above its pIs, a protein will move towards the anode during electrophoresis. At pH values below their pI and –vely charged above their isoelectric points, Thus, at pH values below the pI of a particular protein, it will migrate towards the cathode during electrophoresis. A protein at its isoelectric point will not move in an electric field. ASHISH KUMAR SWAIN 38
- 39. ISOELECTRIC FOCUSING (IEF) ASHISHKUMAR SWAIN 39
- 40. TWO-DIMENSIONAL GEL ELECTROPHORESIS Electrophoresis of all cellular proteins through an SDS gel can separate proteins having a relatively large difference in molecular mass, another physical characteristics must be exploited. In two dimensional electrophoresis, proteins are separated in two sequential steps: First by their charge & then by their mass. In the first step, a cell extract fully denatured by high concentrations (8M) of Urea & then layered on a glass tube filled with polyacrylamide that is saturated with a solution of ampholytes, a mixture of Polyanionic & Polycationic molecules. When placed in electric field, the ampholytes will separate and form a continuous gradient based on their net charge. The most highly polyanionic ampholytes will collect at other end by establishing a pH gradient & the complete experiment involves the basis of protein separation to their pIs by IEF. ASHISH KUMAR SWAIN 40
- 41. TWO-DIMENSIONAL GEL ELECTROPHORESIS ASHISHKUMAR SWAIN 41
- 42. REFERENCES:- Alberts Bet al (2008), Molecular Biology of the cell, 5th ed. Garland Science Publishing. Berg JM, Tymoczko JL and Stryer L (2007), Biochemistry, 6th ed. W.H. Freeman & Company. Garfin DE (2003), Two-Dimensional Gel Electrophoresis: An overview. Trends in Analytical Chemistry 22 263-272 Hames BD (1998), Gel Electrophoresis of Proteins: A Practical Approach, 3rd ed. Oxford University Press, New York. Holler FJ, Crouch SR (2014), Fundamentals of Analytical Chemistry, 9th ed. Cengage Learning. Kumar P. (2018) Fundamentals & Techniques of Biophysics & Molecular Biology, 2nd ed, 17-30, Pathfinder Publication, New Delhi Wilson K, Walker J (2010) Principles and Techniques of Biochemistry and Molecular Biology, 7th ed. Cambridge University Press, New York ASHISH KUMAR SWAIN 42
- 43. THANK YOU E-mail- ashishswain2015@gmail.com Slideshare:-https://www.slideshare.net/AshishKumarSwain5 ASHISH KUMAR SWAIN 43