Electrophoresis Class.

1. Electrophoresis
Principles & Types

2. Each animal/plant/microbe species is composed of different proteins at
varying level/DNA with different base composition. So the techniques
that separate proteins/DNA may help to identify species.
Electrophoresis : The term electrophoresis comes from the Greek, and
means, " transport by electricity“.
In 1807, a Russian Physicist, Alexander Reuss observed a novel
phenomenon - when electricity was passed through a glass tube
containing water and clay, colloidal particles moved towards the positive
electrode. Thus electrophoresis describes the migration of a charged
particle under the influence of an electric field.
In 1955, Oliver Smithies found that separation of human tissue extracts
with high resolution by starch gel electrophoresis.

4. Biological molecules exist in a solution as electrically charged particles at a given pH.
anionic (+vely charged/basic) " Zwitterions "
cationic (-vely charged / acidic) or "amphoteric molecules "
pH greatly influences the total charge of molecules.
When electricity is applied to the medium containing biological molecules, depending on
their net charge & molecular size, they migrate differentially, thus different proteins/DNA
can be separated.
Size of DNA 1 base pair = 660 Dalton; 1kb = 6.6 x 105D = 660kD. DNA packed inside a
human cell nucleus is 15m; if fully stretched ~ 2 meter long. Complete turn of helix is 34
A long and contains 10 nucleotides and diameter of the helix is ~ 20 A. DNA is
negatively charged due to the ionisable phosphate groups and migrate towards the
anode; hence can be separated only based on size and shape.
Total DNA bp (Human) 3.43 x 109 bp = 3400 mega bp (mbp) = 3.5pg (C value); 1pg = 0.98 x
106 kbp = 6.02 x 1011 daltons; functional genes ~42000; average gene size 10 – 15 kb
Proteins: 1 amino acid = 110 Dalton; 1000 amino acids = 110kD.
Principle

6. General Principles
 An idealized, simplified situation: an isolated charged
particle in a non conducting medium.
 The force experienced by a particle in an electrical
field is given by Coulomb’s law,
 F = ZeE (E-electric field: potential per unit length)
 The viscous resistance of the medium to the motion: -
fv (f: the frictional factor)
 The viscous resistance of the medium just balances
the driving force.
fv = F = ZeE

7.  Electrophoretic mobility U (the ratio of velocity to the
strength of the driving field)
U = v/E = Ze/f
 The zonal techniques: In these methods, a thin layer or
zone of the macromolecule solution is electrophoresed
through some kind of matrix.
The kind of supporting matrix used depends on the
type of molecules to be separated and on the
desired basis for separation: charge, molecular
weight, or both.

8. The velocity (v) of charged molecule in an electric field-
v = Eq/F
where F = frictional coefficient, which depends
upon the mass and shape of the molecule.
E = electric field (V/ cm)
q = the net charge on molecule
v = velocity of the molecule.

9.  Almost all electrophoresis of biological macromolecules is at
present carried out on either polyacrylamide or agarose gels
 The matrix provides stability against convection and diffusion. In
addition, in many cases the matrix acts as a molecular sieve to
aid in the separation of molecules on the basis of size.
 allows a permanent record of results through staining after run

14. Interrelation of Resistance, Voltage, Current
and Power
 Two basic electrical equations are important in electrophoresis
 The first is Ohm's Law, I = V/R
 The second is H = VI
 ( heat produced per unit time)
 This can also be expressed as H = I2R
 In electrophoresis, one electrical parameter, either current,
voltage, or power, is always held constant
Ideally voltage is kept constant.
WHY?

15.  Under constant current conditions (velocity is
directly proportional to current), the velocity of
the molecules is maintained, but heat is
generated.
 Under constant voltage conditions, the velocity
slows, but no additional heat is generated
during the course of the run
 Under constant power conditions, the velocity
slows but heating is kept constant

16. Temperature and
Electrophoresis
Important at every stage of electrophoresis
 During Polymerization
- Exothermic Reaction
-Gel irregularities
-Pore size
 During Electrophoresis
-Denaturation of proteins
-Smile effect
-Temperature Regulation of Buffers

17. Effect of matrix concentration

18. Agarose (%)
Range of separation of linear DNA
(in kilobases)
0.3 60 - 5
0.6 20 - 1
0.7 10 - 0.8
0.9 7 - 0.5
1.2 6 - 0.4
1.5 4 - 0.2
2.0 3 - 0.1

19. Based on Buffer System
Based on Support media
Types of electrophoresis

20. Continuous buffer System :-
Most commonly used
Same buffer used in support media and in electrode
chamber
Separation purely molecular size and electrical charge is used
only to induce movement.
e.g.: TBE, TCE, TME, TAE
Based on Buffer System

21. Discontinuous / Multiphasic buffer System:-
Mostly used for proteins
Different buffers used in electrode chamber and in support media.
Proteins enter the gel as a narrow zone - Separating and stacking gel
buffer contain highly electronegative chloride ions. Tank Buffer contains
less electronegative glycine.The pI (Zwitter ions formation) of glycinate
ions is around pH 6.8 (the stacking gel pH). Mobility of glycinate ions is
retarded at this pH. Highly electronegative chlorine will be the leading ion.
Mobility of the sample in the stacking gel is like a sandwich between the
leading chloride and the trailing glycine hence narrow bands & sharp
resolution.
e.g.: TG, LB

22. Chemical nature inert
Availability easy
Electrical conductivity high
Adsorptivity low
Sieving effect desirable
Porosity controlled
Transparency high
Electro-endosmosis (EEO) low
Rigidity moderate to high
Preservation feasible
Toxicity low
Preparation easy
II Based on Support media
Properties:
Different types are: Starch gel, PAGE, Agarose, Paper, Cellulose Acetate

23. TYPES OF ELECTROPHORESIS
1) Zone Electrophoresis
a) Paper Electrophoresis
b) Gel Electrophoresis
c) Thin Layer Electrophoresis
d) Cellulose acetate Electrophoresis
2) Moving Boundary Electrophoresis
a) Capillary Electrophoresis
b) Isotachophoresis
c) Isoelectric Focussing
d) Immuno Electrophoresis

24. Gel Electrophoresis
 Separation is brought about through molecular sieving technique, based on the molecular
size of the substances. Gel material acts as a "molecular sieve”.
 Gel is a colloid in a solid form (99% is water).
 It is important that the support media is electrically neutral.
 Different types of gels which can be used are; Agar and Agarose gel, Starch,
Sephadex, Polyacrylamide gels.
 A porous gel acts as a sieve by retarding or, in some cases, by completely
obstructing the movement of macromolecules while allowing smaller molecules to
migrate freely.
https://youtu.be/lCnge-2yPi0

25. Agar Electrophoresis
Mixture of agarose and agaropectin obtained from seaweeds.
Dissolves in water on heating & forms gel while cooling down about 40C.
Contains negatively charged ions - sulphates & pyruvates; these are
surrounded by counter ions and water which tend to move towards the
cathode during electrophoresis. This backflow is called electroendosmosis
(EEO) which is generally a nuisance and retards the anodal movement of the
molecules.
Can be prepared in various thickness
Transparent & easy to handle
Poor resolution due to EEO
No sieving effect; but molecules move based on their net charges
Can be dried & preserved after staining.

26. Purified form of agar
Polysaccharide with repeating 1,3  D galactopyranose and 1,4-3, 6-
unhydro L- galactopyranose residues obtained from agar
Unlike agar no EEO
Mostly used for DNA & RNA at low agarose concentration
Proteins up to 50 million daltons & above can easily pass through
without hindrance. Hence, protein electrophoresis is based on net
charge differences only in agarose.
DNA molecules are 6 times larger than proteins. Average pore size in
agarose is larger than PAGE/starch hence used for DNA
Usual concentration 0.5% - 3.0%.
Fragile, used in horizontal slab arrangement.
Agarose gel electrophoresis

27. 1.Native PAGE
Acrylamide monomer ((CH2= CH CO NH2) is co-polymerized with cross-
linking agent- N N' methylene bisacrylamide in the presence of an initiator
(ammonium per sulphate) 0.1 to 0.3% w/v and catalyst, tetra methylene
ethylenediamine (TEMED)) .
Gelation occurs due to vinyl polymerization
Relative proportion of monomer & cross-linker decides percentage
of acrylamide & porosity
Used up to 3-30% concentration (pH range=4.0-9.0). Lower
concentration for DNA separation & higher concentration for protein
separation.
High degree of reproducibility & precise porosity
Transparent, no endosmosis, do not absorb UV; suitable for
histochemical analysis.
Polyacrylamide gel electrophoresis (PAGE)

28. AGAR AND 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.
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.

29. Gel Structure of Agarose:
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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.

30. Protein samples heated with detergent SDS and disulfide reducing agent
-mercaptoethanol
Disrupts secondary( Hydrogen bonds), tertiary and quaternary structure
leaving the molecule to produce polypeptide chain in a random coil / “ rod
shaped structure”, imparts an overall –ve charge; charge SS-bands
reduced to SH.
Electrophoresis based on molecular size:
larger molecules - migrate slower
smaller molecules - migrate faster
Molecular weight of polypeptides can be determined.
Used for functional analysis of polypeptides
2. Denaturing PAGE

31. It is prepared by polymerizing acrylamide 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.
ADVANTAGES:
 Gels are stable over wide range of pH and temperature.
 Gels of different pore size can be formed.
 Simple and separation speed is good comparatively.

32. PAGE can be classified according the separation
conditions into:
 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.
 DENATURED-PAGE OR SDS-PAGE:
 Separation is based upon the molecular weight of
proteins.
 The common method for determining MW of proteins.
 Very useful for checking purity of protein samples.

33. PAGE-Procedure
 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.

34. Polyacrylamide Gel Electrophoresis (PAGE)
a) The gel is poured vertically between two
glass plates.
b.) Protein bands are separated on the basis
of relative molecular weight and visualized
with stains.
SLAB PAGE
PAGE PROCEDURE

35. SLAB PAGE
 The Polyacrylamide gel is cast as thin rectangular slab inside a plastic frame and this slab is
placed vertically on a buffer solution taken in a reservoir.
 Several samples dissolved in dense sucrose solution or glycerol are placed in separate
wells cut in to the upper edge of the slab and are covered by the same buffer solution.
Cathode and anode are above and below to produce electric field effect. Different
components migrate simultaneously down parallel lanes in the slab and get separated into
bands.
VISUALIZATION
 After the electrophoresis is complete, the molecules in the gel can be stained to make them
visible.
 Ethidium bromide, silver, or SYBR stain may be used for this process.
 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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36. DC Battery
A/C rectified to DC for prolonged supply
Constant voltage (150 V; 30m A)
Constant Current ( 30m A / gel – usually for TG/LB)
Apparatus to be kept in fridge, to remove heat generated
Source of current

37. Buffer pH value
Phosphate buffer around 7.0
Tris-Borate-EDTA buffer (TBE) around 8.0
Tris-Acetate EDTA buffer (TAE) above 8.0
Tris Glycine buffer (TG) more than 8.5
Tris -Citrate-EDTA buffer (TCE) around 7.0
Tris -EDTA buffer (TE) around 8.0
Tris -Maleic acid -EDTA buffer (TME) around 7.5
Lithium Borate - buffer (LB) around 8.6
Buffers
Weak acid & one of its salts
Resists changes in H+ and OH- ion concentrations & maintains
constant pH
Common buffers:

38. 41
Moving Boundary Electrophoresis
PRINCIPLE:
The moving boundary method allows the charged species to migrate in a free moving
solution without the supporting medium.
INSTRUMENTATION:
o Consists of a U shaped glass cell of rectangular cross section, with electrodes placed on
the one each of the limbs of the cell.
o Sample solution is introduced at the bottom or through the side arm, and the apparatus is
placed in a constant temp. bath at 40o C.
o Detection is done by measuring refractive index throughout the solution.(Schlieren optical
system).

39. 42
ADVANTAGES:
 Biologically active fractions can be recovered without the use of
denaturing agents.
 A reference method for measuring electrophoretic mobilities.
 Minute concentrations of the sample can be detected.(0.05mg/ml by
Interferometric optical system).
DISADVANTAGES:
 Costlier.
 Elaborate optical system are required.
APPLICATION:
 To study homogenecity of a macromolecular system.
 Analysis of complex biological mixtures.

40. 43
Capillary Electrophoresis
• The principle behind electrophoresis is that charged molecules will migrate toward the
opposite pole and separate from each other based on physical characteristics.
• Capillary electrophoresis has grown to become a collection of a range of separation
techniques which involve the application of high voltages across buffer filled capillaries to
achieve separations .
• Capillary electrophoresis, then, is the technique of performing electrophoresis in
buffer-filled, narrow-bore capillaries, normally from 25 to 100 mm in internal diameter
(ID).
• A high voltage (typically 10-30 kV) is applied.
• Capillaries are typically of 50 µm inner diameter and 0.5 to 1 m in length.
• Due to electroosmotic flow, all sample components migrate towards the negative electrode.
• The capillary can also be filled with a gel, which eliminates the electroosmotic flow.
Separation is accomplished as in conventional gel electrophoresis but the capillary allows
higher resolution, greater sensitivity, and on-line detection.
• The capillary is filled with electrolyte solution which conducts current through the inside
of the capillary. The ends of the capillary are dipped into reservoirs filled with the
electrolyte.
• Electrodes (platinum) are inserted into the electrolyte reservoirs to complete the

41. 44
Sample application is done by either
a)High voltage injection-potential is applied causing the sample to enter capillary by
combination of ionic attraction and electroosmotic flow.
b)Pressure injection-pressure difference is used to drive the sample into capillary by
applying vaccum.
• When PD is applied net migration occurs in the direction of cathode.
• Even substance with net negative charge migrate in the direction of cathode due to
the phenomenon called as Electro Osmotic Flow.
• Neutral molecule moves at the same speed as the EOF. Positively charged species
move faster, speed is sum of EOF and Electrophoretic mobility. Negatively charged
molecules lag behind.

42. ELECTROOSMOTIC FLOW
The surface of the silicate glass capillary contains negatively-charged functional groups that attract positively-
charged counterions. The positively-charged ions migrate towards the negative electrode and carry solvent
molecules in the same direction. This overall solvent movement is called electroosmotic flow. During a separation,
uncharged molecules move at the same velocity as the electroosmotic flow (with very little separation). Positively-
charged ions move faster and negatively-charged ions move slower.
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43. • A small volume of sample is moved into one end of the capillary. The capillary passes
through a detector, usually a UV absorbance detector, at the opposite end of the
capillary.
• Application of a voltage causes movement of sample ions towards their appropriate
electrode usually passing through the detector.
• A plot of detector response with time is generated which is termed an
electropherogram.
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44. 47
Isotachophoresis
The technique of isotachophoresis depends on the development of potential gradient.
PRINCIPLE:
 Based on principle of moving boundary electrophoresis.
 A leading electrolyte(e.g. chloride) with a higher mobility than the analytes, and a trailing
electrolyte(e.g. glycinate) with a lower mobility are used.
 Solution in which the separation takes place is normally an aqueous medium, which contains sucrose to
provide a higher density to the solution.
 Where the separation by Isoelectric focusing depends on the existence of a pH gradient in the
system. The technique of Isotachophoresis depends on the development of a potential gradient.
 Separation of the ionic components of the sample is achieved through stacking them into discrete zones
in order of their mobilities, producing very high resolution.
In the example shown here
three particle classes with
different charges are being
separated and preconcentrated
via electrophoresis.
After the separation is
concluded all particles move at a
constant speed

45. 48
We fill one well with slow
trailing electrolyte (T) mixed
with samples (S1,S2), and the
other well with fast leading
electrolyte (L). When we apply
an electric field, ions
electromigrate through a
microchannel according to their
electrophoretic mobilities.
Sample ions overspeed the slow
trailing electrolyte, but cannot
overspeed the fast leading
electrolyte ;
consequently, they focus at
the interface.
Sample continues to
accumulate. If sample
concentration approaches
the concentration of the
leading electrolyte, samples
self-segregate into discrete
zones.

46. 49
 The analytes are positioned between the electrolytes and, when the voltage is applied, they migrate in
order of decreasing mobility.
 This establishes the potential gradient; from that point on, all the analytes move at the same speed.
 Individual zones border one another but represent completely separated components with out overlap.
 In isotachophoresis no background electrolyte(buffer) is mixed with the sample, so current flow is carried
only by charged sample ions.
 Once a faster moving component separates completely from a slower moving one, It creates a
region of depleted charge between the two that increases the resistance and therefore local
voltage in that region.
 This increased voltage causes the slower component to migrate faster and close the gap, thereby
concentrating it and increasing the conductivity of its zone until it matches that of the faster ion.
 Ultimately all ions migration at the rate of the faster ion in the zones that differ in thickness, depending on
their original concentrations.
APPLICATION:
 Isotachophoresis that been used for the separation of proteins as well as
inorganic substances.

47. Makes use the principle of pI (isoelectric point)
Media with pH gradient
Strong acid at anode and strong base at Cathode
pH gradient achieved with commercially available synthetic poly
ampholytes/ampholenes (MW 300-600).
Pre run required for 15 min
Using PAGE with large pore size
Samples can be applied anywhere over the gel
High voltage 2500 V used ( at 80 C.)
Ultra thin (0.1 mm thick) PAGE for separation of crystalline, hemoglobin,
myoglobin.
High resolution can be achieved permitting separation of proteins differing only
by 0.01 pI
Isoelectric Focusing (IEF)

48. Technique of IEF & SDS PAGE combined
For fine separation of polypeptides having only minute
differences in pI & mol.wt
First separation by IEF
Next separation according to mol. wt (SDS- PAGE) which
separates protein according to size at right angles to the
direction of 1st separation.
Series of spots formed in gel.
Two - dimensional (2D) electrophoresis

49.  In the first dimension, proteins are resolved in according to their isoelectric points (PI) using
immobilized pH gradient electrophoresis (IPGE), isoelectric focusing (IEF), or non-equilibrium pH
gradient electrophoresis. (Horizontal separation)
 In the second dimension, proteins are separated according to their approximate molecular weight using
SDS-PAGE. (Vertical separation).
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50. Continuous and Discontinuous Buffer
Systems
 A continuous system has only a single separating gel and uses
the same buffer in the tanks and the gel
 In a discontinuous system a nonrestrictive large pore gel, called a
stacking gel, is layered on top of a separating gel
 The resolution obtainable in a discontinuous system is much
greater than that obtainable in a continuous one. However, the
continuous system is a little easier to set up.

52. What is EEO & why low???

53. Electroendosmosis cont

57. Electrophoresis Equipment: Horizontal or
Submarine Gel
DNA/RNA is negatively charged: RUN TO RED

58. Agarose Gel Electrophoresis System

59. Proteins
General – Coomassie brilliant blue R, Kenacid blue, Amido
black.
Specific – Oil red O, PAS, Rubeanic acid, Transferrin-specific &
for calcium binding proteins
Steps fixing
staining
destaining
Allozymes Histochemical staining
DNA/RNA EtBr, SyBR stains, Propidium iodide and
silver staining
Staining Systems

60. Images of different types of gel electrophoresis
RAPD pattern of fish DNA with Operon primer
Agarose (1.5%) electrophoresis
Microsatellite pattern of fish DNA in PAGE
with silver staining.
Allozyme (SOD) pattern in PAGE
Allozyme (Esterase) pattern in PAGE Ultra-thin IEF of fish haemoglobin
2D gel electrophoresis of frog oocytes
(IEF and SDS PAGE at right angles)