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.

1. GUIDED BY:-
DR. ANIL KUMAR
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
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2. CONTENTS (as per Syllabus)
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.
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3. INTRODUCTION
 Electrophoresis is a 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.
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4. MECHANISM OF ELECTROPHORESIS
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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
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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
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7. TYPES OF ELECTROPHORESIS
ELECTROPHORESIS
MOVING BOUNDARY
ELECROPHORESIS
Capilary Electrophoresis
Isotachophoresis
Isoelectric Focusing
Immunoelectrophoresis
ZONE
ELECTROPHORESIS
Paper Electrophoresis
Thin Layer Electrophoresis
Gel Electrophoresis
Cellulose Acetate
Electrophoresis
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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.
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9. ZONE ELECTROPHORESIS
 It is 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.
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10. GEL ELECTROPHORESIS
 Gel electrophoresis 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.
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11. TOOLS & PROCEDURES OF GEL ELECTROPHORESIS
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12. METHODS OF GEL ELECTROPHORESIS
 Gel Electrophoresis is carried out in
two methods :
1. Horizontal gel electrophoresis.
2. Vertical gel electrophoresis
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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.
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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.
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15. TYPES OF GEL ELECTROPHORESIS
GEL ELECTROPHORESIS
AGAROSE GEL
ELECTROPHORESIS
STARCH GEL
ELECTROPHORESIS
POLYACRYLAMIDE GEL
ELECTROPHORESIS
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16. TYPES OF GEL
AGAROSE GEL
 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.
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17. AGAR & AGAROSE GEL
 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.
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18. STRUCTURE OF AGAR
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19. STRUCTURE OF AGAROSE GEL
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20. ADVANTAGES:
 Easy to prepare 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.
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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.
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22. POLYACRYLAMIDE GEL
ELECTROPHORESIS
 It is 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.
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23. ASHISH KUMAR SWAIN
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24. PAGE PROCEDURES
 The gel 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.
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25. EXPERIMENTATION STEPS
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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.
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27. TYPES OF PAGE
PAGE
SDS-PAGE
NATIVE
PAGE
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28. TYPES OF PAGE
NATIVE PAGE
 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.
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29. NATIVE-PAGE
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30. SDS-PAGE
 SDS-PAGE, sodium dodecyl 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.
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31. SODIUM DODECYL SULFATE (SDS)
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32. SDS-PAGE
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33. SDS-PAGE
BEFORE SDS AFTER SDS
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34. PAGE APPARATUS
SDS-coated large proteins 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
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35. ADVANTAGES
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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
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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.
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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.
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39. ISOELECTRIC FOCUSING (IEF)
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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.
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41. TWO-DIMENSIONAL GEL ELECTROPHORESIS
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42. REFERENCES:-
 Alberts B et 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
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43. THANK YOU
E-mail- ashishswain2015@gmail.com
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