Electrophoresis

Electrophoresis is the movement of charged particles through an electrode when subjected to an electric Field
Cations move towards cathode
Anions move towards anode
By this technique solutes are separated by their different rates of travel through an electric field.
Commonly used in biological analysis, particularly in the separations of proteins, peptides and nucleic acids

1. Tapeshwar Yadav (Lecturer) BMLT, DNHE, M.Sc. Medical Biochemistry ELECTROPHORESIS

2. Introduction Electrophoresis is the movement of charged particles through an electrode when subjected to an electric Field Cations move towards cathode Anions move towards anode By this technique solutes are separated by their different rates of travel through an electric field. Commonly used in biological analysis, particularly in the separations of proteins, peptides and nucleic acids

3. 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 .

4. Factors affecting Electrophoresis The rate of migration of a solute in an electric field depends on the following factors- 1) Net charge on the particle 2) Mass and shape of the particles 3) p H of the medium 4) Strength of electric field 5) Properties of supporting medium 6) Temperature

5. Electrophoretic Mobility Electrophoretic mobility is defined as the rate of migration (cm/sec) per unit field strength(Volts/cm) µ=Q/6 π r η Where µ- Electrophoretic mobility Q-Net charge on the ion r- Ionic radius of the solute η - Viscosity of the medium

6. Electrophoretic mobility The Electrophoretic mobility is directly proportional to net charge and inversely proportional to molecular size and viscosity of the electrophoresis medium The p H of solution affects the mobility of the ion by determining the amount and nature of charge Proteins, nucleic acids, nucleotides and amino acids bear charged polar groups making them suitable groups for electrophoresis

7. Electrophoretic mobility Carbohydrates carrying no charged groups are first bound to charged groups like Borate or Sulfite ions and then electrophoresis is carried out. Lipids are not electrophoresed because electrophoretic current requires polar solvents in which most lipids are insoluble

8. Electrophoresis Apparatus Electrophoresis apparatus consists of- Buffer tank -to hold the buffer Buffer Electrodes- made of platinum or carbon Power supply Support media Note- Choice of buffer depends on the nature of substance to be separated and the electricity is supplied at a constant current and voltage.

9. Electrophoresis Apparatus The electrophoresis support on which separation takes place may contact the buffer directly or by means of wicks. The entire apparatus is covered to minimize separation

10. Support media for electrophoresis 1) Filter Paper 2) Cellulose acetate membrane 3) Agar or Agarose gel 4) Starch Gel 5) Polyacrylamide gel

11. Buffers: The buffer serves as a multifunctional component in the electrophoretic process as it, Carriers the applied current. Establishes the pH at which electrophoresis is performed and Determines the Electrical charge on the solute .

12. As the ionic strength of the buffer increases, the proportion of current carried by the buffer will increase and the share of the current carried by the sample will decrease, thus slowing down the rate of migration. So always ionic strength of 0.05M is preferred for the separation of proteins, or lipoproteins in an electric field.

13. Support Media The Support Medium provides the matrix in which separation takes place. Various types of support media are used in electrophoresis and vary from pure buffer solution in a capillary to insoluble gels (e.g., sheet, slabs, or columns of starch, agarose, or polyacrylamide) or membrances of cellulose acetate.

14. Working procedure of electrophoresis: General Operations performed in conventional electrophoresis include Separation , Staining Detection , and Quantification .

15. Working procedure of Electrophoresis The porous support is hydrated and placed between the two chambers containing a suitable buffer Sample is applied (in microlitres) on the support at the cathode end and the components are allowed to move from cathode to anode under the influence of direct current. At the end of the run, the support is removed and the position of the molecules on the support is fixed with a fixative to prevent sample diffusion.

16. Contd… The separated components are then stained to visualize them. The bands can be quantitated (by elution or by scanning with a densitometer) as the uptake of the dye is directly proportional to the concentration of the molecule in each band.

17. 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 c) Isoelectric Focussing d) Immuno Electrophoresis

18. 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

19. APPLICATIONS OF ELECTROPHORESIS 7. 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. 8. Electrophoresis in combination with autoradiography is used to study the binding of iron to serum proteins . 9. Used for analysis of terpenoids , steroids and antibiotics. 10. For testing purity of thyroid hormones by zone electrophoresis.

20. APPLICATIONS OF ELECTROPHORESIS 11. Paper chromato -electrophoresis is used to separate free Insulin from plasma proteins. 12. It is used for diagnosis of various diseases of kidney , liver and CVS. 13. It is also used for separation of Scopolamine and Ephedrine using buffer at PH 4.2. 14. Electrophoresis is also used for separation of carbohydrates and vitamins. 15. Quantitative separation of all fractions of cellular entities, antibiotics, RBC, Enzymes etc is possible.

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