Electrophoresis

introduction and types and its properties

1. FACILITATED TO – SRINIVAS RAO SIR ASSISTANT PROFESSOR ( PH. ANALYSIS ) SUBMITTED BY- SANTHOSH KUMAR T S M.PHARM 1 ST YEAR PRESENTATION ON : ELECTROPHORESIS KARNATAKA COLLEGE OF PHARMACY BANGALORE

2. CONTENTS Introduction about electrophoresis Principle Theory Electrophoretic Techniques Applications Types of electrophoresis

3. Introduced by ARNE TISELIUS in 1937, for the separation of proteins. Defined mainly as the migration of charged molecules under the influence of an external magnetic field. This method is used for the separation of A) Proteins B) Nucleic acid C) Polysaccharides INTRODUCTION

4. Electrophoresis is a physical method of analysis which involves separation of the compounds that are capable of acquiring electric charge in conducting electrodes. Electrophoresis may be defined as the migration of the charged particle through a solution under the influence of an external electrical field. Ions that are suspended between two electrodes tends to travel towards the electrodes that bears opposite charges.

5. PURPOSE FOR CARRYING OUT ELECTROPHORESIS 1. To determine the number, amount and mobility of components in a given sample or to separate them. 2. To obtain information about the electrical double layers surrounding the particles. 3. Determination of molecular weight of proteins and DNA sequencing

6. PRINCIPLE Any charged ion or molecule migrates when placed in an electric field. The rate of migration depend upon its net charge, size, shape and the applied electric current.Rate of migration (separation) depends upon e/m (charge to mass) ratio The migration of particles or the rate of travel of particle, in electrophoretic system depends on properties of the particles as well as the instrumental system Characteristic of particles Property of electric field Temperature Nature of suspended medium

7. ELECTROPHORETIC MOBILITY It is defined as the rate of migration (cm/sec) per unit field strength (volts/cm) . Mobility of particle is calculated by Strokes law :- μ = Q/6 π r η where Q = charge on the particles μ = mobility of particle r = radius η = viscosity of the medium

8. FACTORS AFFECTING ELECTROPHORETIC MOBILITY 1 Charge – higher the charge greater the electrophoretic mobility. 2 Size – bigger the molecule greater are the frictional and electrostatic forces exerted on it by the medium. Consequently, larger particles have smaller electrophoretic mobility compared to smaller particles. 3. Shape – rounded contours elicit lesser frictional and electrostatic retardation compared to sharp contours. Therefore globular protein move faster than fibrous protein.

9. THEORY Migration of charged particles on potential gradient and space in between 2 electrodes. Hence field strength (x) = E/S Where, E = Potential gradient applied S = space between 2 electrodes basically electrophoresis is an a separation technique Simple, rapid and highly sensitive used in clinical laboratories to separate charged molecules from each other in presence of electric field Proteins in body fluids: serum, urine, CSF Proteins in erythrocytes: hemoglobin Nucleic acids: DNA, RNA

10. The rate of migration of an ion in electrical field depend on factors, 1. Net charge of molecule 2. mobility 3. Size and shape of particle 4. Strength of electrical field 5. Properties of supporting medium 6. Temperature of operation

11. 1. Mobility Under the electrical field, the mobility of the particle is determined by two factors: Its charge Frictional coefficient 2. Size and shape Size and shape of the particle decide the velocity with which the particle will migrate under the given electrical field and the medium

12. 3. Strength of electrical field It determined by the force exerted on the particle, and the charge the particle carrying. F=QV when force is exerted on the particle it start moving, however the moment is restricted by the experience of the frictional force because of the viscosity.

13. 4. Supporting medium Supporting medium is an matrix in which the protein separation takes place. Various type has been used for the separation either on slab or capillary form. Separation is based on to the charge to mass ratio of protein depending on the pore size of the medium, possibly the molecular size. Starch gel Cellulose acetate Agarose Polyacrylamide gel.

14. 1. Agarose Gel A linear polysaccharide (made-up of repeat unit of agarobiose -alternating unit of galactose and 3,6-anhydrogalactose). Used in conc as 1% and 3%. Pore size is controlled by the % of agarose used. It can be stored for longer duration. 2. Cellulose acetate Thermoplastic resin made by treating cellulose with acetic anhydride to acetylate the hydroxyl group. As the film is soak in buffer, the space are filled. Because of their opacity, the film has to be made transparent by soaking in 95:5 methanol:glacial acetic acid.

15. Polyacrylamide Frequently referred to as PAGE. They are defined in terms of total percentage of acrylamide present, and pore size vary with conc. Made in conc between 3-30% acrylamide . Proteins are separated on the basis of charge to mass ratio and molecular size, a phenomenon called Molecular sieving .

16. 4. Effect of pH on Mobility As the molecule exist as amphoteric , they will carry the charges based on the solvent pH. Their overall net charge is NEUTRAL when it is at zwitter ion state. And hence the mobility is retarded to zero. Mobility is directly proportional to the magnitude of the charge, which is functional of the pH of solvent. The pH is maintained by the use of Buffers of different pH.

17. ELECTROPHORETIC TECHNIQUES

18. Electrophoresis Separation When performed on precast or agarose gel, following steps are followed; Excess buffer removed 5-7 μ L sample Placed in electrode chamber Current application Gel is rinsed, fixed and dried Stained Scanned under densitometry

19. STAINING Protein is precipitated in gel by using acetic acid or methanol ( this will prevent the diffusion of protein out of the gel when submerged in staining sloution )

20. Different stains of Electrophoresis Plasma Proteins 1 Amido black 2 Coomassie Brilliant Blue 3 Bromophenol Blue Hemoglobins 1 Amido black 2 Coomassie Brilliant Blue 3 Ponceau Red Lipoproteins Sudan Black DNA ( Fluorescent dyes) 1 Ethidium Bromide 2 Sybr Green, Sybr Gold

21. 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 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.

22. 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.

23. Clinical applications of Electrophoresis Serum Protein Electrophoresis Lipoprotein Analysis Diagnosis of Haemoglobinopathies and Haemoglobin A1c Determination of Serum Protein Phenotypes and Micro heterogeneities eg . α1- antitrypsin deficiency, MM Genotyping of Proteins eg . ApoE analysis for Alzheimer’s disease (polymorphic protein)

24. TYPES OF ELECTROPHORESIS 1) Zone Electrophoresis Paper Electrophoresis Gel Electrophoresis Thin Layer Electrophoresis Cellulose acetate Electrophoresis 2) Moving Boundary Electrophoresis Capillary Electrophoresis Isotachophoresis Isoelectric Focussing Immuno Electrophoresis

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27. The buffer in electrophoresis has two fold purpose: 1 Carry applied electrical current 2 They set the pH as which electrophoresis is carried out. Thus they determine; 1 Type of charge on solute. 2 Extent of ionization of solute 3 Electrode towards which the solute will migrate. 4 The buffer ionic strength will determine the thickness of the ionic cloud Commonly buffers used; 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

28. 32 Solution condition The solution conditions are important variables. For eg . An acidic pH would favors protonation of basic centers of a protein, producing a – ve charged molecule. It is not desirable to choose a pH such that the protein is at its isoelectric point and exists as the uncharged Zwitterions( a species not mobile in the imposed electrical field)

29. 33 Ionic strength Electrophoretic mobility decreases with the supporting electrolyte ionic strength Generally, the ionic strengths employed in electrophoresis range from 0.01 to 0.10. The temperature of the solution is important because the solution viscosity varies with temperature and the mobility inc. with temperature Temperature