* 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.
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.
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.
Types of electrophoresis 1. Based on Buffer System 2. Based on Support media
I Based on Buffer System •Continuous buffer System :- * Most commonly used * Same buffer used in support media and in electrodechamber * Separation purely molecular size and electrical charge is used only to induce movement. e.g.: TBE, TCE, TME, TAE
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
II Based on Support mediaProperties: 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 easyDifferent types are: Starch gel, PAGE, Agarose, Paper, Cellulose Acetate
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
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.
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
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.
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
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.
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
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
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.
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
Buffers * Weak acid & one of its salts * Resists changes in H+ and OH- ion concentrations & maintains constant pH Commonly employed buffers:- Buffer pH valuePhosphate buffer around 7.0Tris-Borate-EDTA buffer (TBE) around 8.0Tris-Acetate EDTA buffer (TAE) above 8.0Tris Glycine buffer (TG) more than 8.5Tris -Citrate-EDTA buffer (TCE) around 7.0Tris -EDTA buffer (TE) around 8.0Tris -Maleic acid -EDTA buffer (TME) around 7.5Lithium Borate - buffer (LB) around 8.6
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.
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.
Staining SystemsProteins General – Coomassie brilliant blue R, Kenacid blue, Amido black. Specific – Oil red O, PAS, Rubeanic acid, Transferrin-specific & for calcium binding proteinsSteps * fixing * staining * destainingAllozymes - Histochemical stainingDNA - EtBr, SyBR green, Propidium iodide and silver staining
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.
Images of different types of gel electrophoresisRAPD 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)