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.
3. Principle
• 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 15µm; 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 no.of 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; no.of functional genes
~42000; average gene size 10 – 15 kb
• Proteins: 1 amino acid = 110 Dalton; 1000 amino acids = 110kD.
4. 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.
6. I Based on Buffer System
•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
7. B. 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
8. II Based on Support media
Properties:
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
Different types are: Starch gel, PAGE, Agarose, Paper, Cellulose Acetate
9. Paper gel electrophoresis
• Historical significance; first media used for electrophoresis by pioneering
investigators like Tiselius.
• Used in clinical investigations of serum and other body fluids.
* Adsorbs proteins
• Poor conductivity
* Background staining
* Hydroxyl group of cellulose bind with proteins and retard electrophoretic
movements causing trailing of bands & poor resolution
* Non-transparent
* Can be stored easily
10. 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.
11. Starch gel electrophoresis
* Introduced by Smithies (1955).
* Starch used as Supporting media.
* 2 forms - α amylose (unbranched) & amylopectin (branched)
polymers.
* Mostly used for protein separation.
* Cooking hydrolyzed potato starch with buffer until a uniform
consistency is achieved.
* Good for proteins.
Problems :
• Degassing is required.
• Less allozyme alleles reported in fish
12. Agarose gel electrophoresis
* Purified form of agar
* Polysaccharide with repeating 1,3 β 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.
13. Cellulose acetate electrophoresis
* Hydroxyl groups of cellulose (paper) converted to acetyl groups-
thus molecules become non-adsorbing
* Non-toxic
* Resolution is poor
* Transparent
* Readily be dissolved in various solvents thus allowing easy
recovery separated components
* Widely used in clinical applications
14. Polyacrylamide gel electrophoresis (PAGE)
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.
15. 2. Denaturing PAGE
* Protein samples heated with detergent SDS and disulfide reducing
agent β 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
16. Isoelectric Focusing (IEF)
* 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
* UsingPage 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 crystallin, haemoglobin,
myoglobin.
* High resolution can be achieved permitting separation of proteins differing
only by 0.01pI
17. Two - dimensional (2D) electrophoresis
* 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.
18. Source of current
* 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
19. Buffers
* Weak acid & one of its salts
* Resists changes in H+ and OH- ion concentrations &
maintains constant pH
Commonly employed buffers:-
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
20. Proteins studied for fish species identification
* Liver extracts (water soluble - enzymes of glycolytic pathway)
* Sarcoplasmic (water soluble) protein located in sarcolemma
(myogen)
* Can be used for inter & intra species differentiation.
* Myofibrillar proteins or salt soluble proteins
* Present in the myofibrils of the muscle fibre
* myosin and tropomyosin
* Myosin-Hexameric protein with 2 identical heavy chains & 4
light chains
* Electrophoresis of light chain fractions of myosin can be
used for species specific reactions
* Tropomyosin- (heat stable). species specific profiles can be
made from fish products also-adulteration in products can
be examined.
* Crystallins- Secreted by only one type of cells in eye;
structural proteins (3 proteins - α, β and γ)
β & γ can be used for resolving taxonomic ambiguities.
21. Allozymes
* Isozymes: functionally similar, separable forms of enzymes
encoded by one or more loci.
* Isozymes of different alleles of same locus-allozymes
* Co-dominant gene products, inherits in Mendelian fashion.
* Widely used for stock identification, species-specific studies.
* Allele frequencies respond to mutation & gene flow.
Drawback:- Functional gene products, low level of polymorphism.
22. Staining Systems
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 - EtBr, SyBR green, Propidium iodide and
silver staining
23. Types of staining systems for enzymes.
• The enzyme being stained for converts the substrate directly into a
visible product.
B. The enzyme that is being stained for converts the substrate into a
product that is not visible, but can be made visible by the addition of
other histochemicals.
C. The enzyme being stained for converts the substrate into a product that
is not visible but can be converted by a linking enzyme into a second
product that can be made visible.
25. Images of different types of gel electrophoresis
RAPD pattern of fish DNA with Operon primer
Microsatellite pattern of fish DNA in PAGE Allozyme (SOD) pattern in PAGE
Agarose (1.5%) electrophoresis
with silver staining.
2D gel electrophoresis of frog oocytes
Allozyme (Esterase) pattern in PAGE Ultra-thin IEF of fish haemoglobin
(IEF and SDS PAGE at right angles)
