ELECTROPHORESIS.pptx

General introduction about electrophoresis

Principle

Working condition of electrophoresis

Factors affecting separation of electrophoresis

Application of electrophoresis

Types of electrophoresis

1. Aditya bangalore institute OF PHARMACY education and research. DEPARTMENT OF PHARMACOLOY MODERN PHARMACEUTICAL ANALYSIS PRESENTATION ON ELECTROPHORESIS PRESENTED BY RAJESH YADAV M PHARM

2. ELECTROPHORESIS General introduction about electrophoresis Principle Working condition of electrophoresis Factors affecting separation of electrophoresis Application of electrophoresis Types of electrophoresis

3. ELECTROPHORESIS INTRODUCTION : Electrophoresis is a physical method of analysis which involves separation of the compounds that are capable of acquiring electric charge in conducting electrodes.

4. ELECTROPHORESIS DEFINITION : 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. ELECTROPHORESIS 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

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7. ELECTROPHORESIS 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

8. ELECTROPHORESIS 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. Can be represented by following eq. v = E * q ----------- f

9. ELECTROPHORESIS v = velocity of migration of the molecule. E = electric field in volts per cm q = net electric charge on the molecule f = frictional coefficient

10. ELECTROPHORESIS The movement of charged particle in an electric field is expressed in terms of electrophoretic mobility ,denoted by µ. where, µ = v/E OR µ = q/f For molecules with similar conformation f varies with size but not with shape. Thus electrophoretic mobility (µ) of a molecule is directly proportional to charge density(charge\mass ratio).

11. 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.

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

13. ELECTROPHORESIS 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

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15. ELECTROPHORESIS 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.

16. ELECTROPHORESIS 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.

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19. ELECTROPHORESIS 2 Power supply Drives the moment of ionic species in the medium and allow the adjustment and control of the current or voltage. Constant delivery is required. Pulsed power can also be applied

20. ELECTROPHORESIS 3 Buffer 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.

21. ELECTROPHORESIS 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 Tris -EDTA buffer (TE) around 8.0 Tris - Maleic acid -EDTA buffer (TME) around 7.5

22. ELECTROPHORESIS 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.

23. ELECTROPHORESIS Starch gel Cellulose acetate Agarose Polyacrylamide gel

24. ELECTROPHORESIS 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%. The gelling property are attributed to both inter- and intramolecular hydrogen bonding Pore size is controlled by the % of agarose used. Large pore size are formed with lower conc and vice versa. Purity of the agarose is based on the number of sulphate conc , lower the conc of sulphate higher is the purity of agarose .

25. ELECTROPHORESIS 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.

26. ELECTROPHORESIS 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.

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28. ELECTROPHORESIS 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. It can be stored for longer duration.

29. ELECTROPHORESIS 3 Polyacrylamide Frequently referred to as PAGE. Cross-linked polyacrylamide gel are formed from the polymerization of the monomer in presence of small amount of N,N”- methylenebisacrylamide . They are defined in terms of total percentage of acrylamide present, and pore size vary with conc. Made in conc between 3-30% acrylamide . Thus low % has large pore size and vice versa. Proteins are separated on the basis of charge to mass ratio and molecular size, a phenomenon called Molecular sieving.

30. ELECTROPHORESIS 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.

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32. ELECTROPHORESIS a. 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

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

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36. ELECTROPHORESIS 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

37. ELECTROPHORESIS 1 ZONE ELECTROPHORESIS It involves the migration of the charged particle on the supporting media. Paper, Cellulose acetate membrane, Starch Gel, Poly acrylamide . Components separated are distributed into discrete zone on the support media. Supporting media is saturated with buffer solution, small volume of the sample is applied as narrow band. On application of PD at the ends of a strip components migrates at a rate determined by its electrophoretic mobility.

38. ELECTROPHORESIS ADVANTAGES: Useful in biochemical investigations. Small quanity of sample can be analysed . Cost is low and easy maintenance. DISADVANTAGES: Unsuitable for accurate mobility and isoelectric point determination. Due to the presence of supporting medium, technical complications such as capillary flow, electro osmosis, adsorption and molecular sieving are introduced

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42. ELECTROPHORESIS Filter paper such as Whatmann no1 and no 3mm in strip of 3 or 5cm wide have been used to good effect. Separation takes place in 12 to 14hrs. ADVANTAGES: It is economical Easy to use.

43. ELECTROPHORESIS DISADVANTAGES: Certain compounds such as proteins, hydrophilic molecules cannot be resolved due to the adsorptive and ionogenic properties of paper which results in tailing and distortion of component bands. Electro osmosis.

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46. ELECTROPHORESIS 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.

47. ELECTROPHORESIS 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

48. ELECTROPHORESIS 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.

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51. ELECTROPHORESIS THIN LAYER ELECTROPHORESIS Studies can be carried out in thin layer of silica, keisulguhr , alumina. ADVANTAGES: Less time consuming and good resolution.

52. ELECTROPHORESIS APPLICATION: Widely used in combined electrophoretic-chromatography studies in two dimensional study of proteins and nucleic acid hydrolysates .

53. ELECTROPHORESIS CELLULOSEACETATE ELECTROPHORESIS It contains 2-3 acetyl groups per glucose unit and its adsorption capacity is less than that of paper. It gives sharper bands. Provides a good background for staining glycoproteins . ADVANTAGE: No tailing of proteins or hydrophilic materials. Available in wide range of particle size and layer thickness. Give sharp bands and offer good resolution. High voltage can be applied which will enhance the resolution.

54. ELECTROPHORESIS DISADVANTAGE: Expensive. Presence of sulphonic and carboxylic residue causes induced electroosmosis during electrophoresis. APPLICATION: Widely used in analysis of clinical and biological protein samples (albumin and globulins). Alternative to paper electrophoresis

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56. ELECTROPHORESIS ADVANTAGES: Biologically active fractions can be recovered without the use of denaturing agents. Minute concentrations of the sample can be detected.(0.05mg/ml by Interferometric optical system). DISADVANTAGES: Costlier. Elaborate optical system are required.

57. ELECTROPHORESIS APPLICATION: To study homogenecity of a macromolecular system. Analysis of complex biological mixtures.

58. ELECTROPHORESIS APPLICATIONS OF ELECTROPHORESIS DNA Sequencing Medical Research Protein research/purification Agricultural testing Separation of organic acid, alkaloids, carbohydrates, amino acids, alcohols, phenols, nucleic acids, insulin. In food industry It is employed in biochemical and clinical fields i.e. in the study of protein mixtures such as blood serum, haemoglobins and in the study of antigen- antibody interactions.

59. ELECTROPHORESIS Electrophoresis in combination with autoradiography is used to study the binding of iron to serum proteins. Used for analysis of terpenoids , steroids and antibiotics. For testing purity of thyroid hormones by zone electrophoresis. Paper chromato -electrophoresis is used to separate free Insulin from plasma proteins. It is used for diagnosis of various diseases of kidney , liver and CVS. It is also used for separation of Scopolamine and Ephedrine using buffer at PH 4.2. Electrophoresis is also used for separation of carbohydrates and vitamins. Quantitative separation of all fractions of cellular entities, antibiotics, RBC, Enzymes etc is possible

60. ELECTROPHORESIS 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)

61. ELECTROPHORESIS Small Molecules (Drugs, Steroids) Monitoring Cerebrospinal Fluid Analysis Urine Analysis ( determination of GNs)

62. ELECTROPHORESIS THANK YOU DO U HAVE ANY QUESTION