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Electrophoresis
- 2. Electrophoresis is themigration of charged particle in an electric field towards the electrode bearing the opposite charge.
- 3. PRINCIPLE Many importantbiological molecules such as aminoacids,peptides,proteins,nucleotides & nucleic acid posses ionizable groups & therefore at any given pH exist in solution as electrically charged species, either as anions (-) or cations (+).Under the influence of an electrical field these charged particles (cations) migrate to the cathode (Negative electrode) or (anions) move to the anode( positive electrode), depending on the nature of their net charge.
- 4. FACTORS AFFECTING ELECTROPHORESIS 1.Net charge on the molecule Grater charge grater mobility 2.Size & shape of molecule Grater size ↓se mobility Shape Globular -----------↑se mobility Fibrous ----------- ↓se mobility
- 5. 3.Ionic strength &pH of buffer ↑ se ionic strength ↑se heat production ↓se mobility ↓se ionic strength less heat production ↑se mobility pH of buffer 1-11 4.Strength of electric field 5.Temprature 6.Nature of support medium
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- 7. ELECTROPHORESIS APPARATUS Electrophoresis tank – Buffer chamber Electrodes Electrical connection to power supply Space for keeping support medium Power tank – to provide constant currents or constant voltage
- 8. ELECTROPHORETIC TANK ANDPOWER PACK
- 9. APPARATUS 1.Electrophoresis iscarried out in a tank suitable for supporting medium e.g. paper or gel. Major part of tank include – Buffer chamber Electrodes Electrical connection to power supply Space for keeping support medium i.e. paper & slides of agar gel. 2.Power pack
- 10. SUPPORT MEDIUM Filterpaper Cellulose acetate membrane Agar & agarose Starch Polyacrylamide
- 11. ELECTROPHORETIC TANK ANDPOWER PACK
- 12. ADDITIONAL REQUIREMENTS Buffer Fixative Staining solution Destaining solution Densitometer – is essentially a double beam filter photometer or spectrophotometer that scans the electrophoretic strip ( in the form of agarose , cellulose acetate or polyacrylamide) as it moves past the optical system.
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- 14. PROCEDURE 1. Sampleto be separated is applied to a supporting medium (paper, cellulose acetate, agar gel, polyacrylamide gel etc.) 2. Electrophoresis is carried out at desired constant voltage or constant current in presence of specific pH. 3. After completion of electrophoresis the supporting medium is placed in a fixative to prevent diffusion of separated fractions. 4. Separated fraction is then visualized by using appropriate stains e.g. Bromophenol Blue & Amino Schwartz for plasma proteins & Sudan Black for lipoproteins. 5. Quantification of each fraction is done by either densitometer or elution followed by colorimeter or spectrophotometer of eluted fraction.
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- 16. APPLICATIONS 1.Separation of biological molecules like plasma proteins , nucleic acids, nucleotides, charged carbohydrate derivatives, glycoproteins, lipoproteins, hemoglobin variants, isoenzymes etc.
- 17. 2.Analytical Applications- Determinationof DNA sequences Southern & Northern Blotting Restriction Mapping of DNA 3. In Protein Study Determination of subunit stoichiometry Determination of molecular weight
- 18. TECHNIQUE APPICATION OFSAMPLE RUNNING OF SAMPLE VISUALISATION OF SAMPLE QUANTIFICATION
- 19. APPLICATIONS Types of electrophoresis Support medium applications paper Whatman’s paper no.1 Separation of peptides Plasma proteins Nucleic acid Charged carbohydrates Cellulose acetate Cellulose acetate strips in which hydroxyl gr.of cellulose is acetate with acetic anhydride Separation of Glycoproteins Lipoproteins Hb derivatives Gel :- Based on charge & molecular size a. Agarose gel Composed of agarose & agaropectin Separation of plasma proteins Lipoproteins Hb derivatives
- 20. CONTINUED b. StarchPartially hydrolysed starch Separation of isoenzymes c. polyacrylamide Polyacrylamide gel layers of varying pore size Separation of Glycoproteins Lipoproteins Hb derivatives DNA fragments Isoelectricfocussing Polyacrylamide gel Separation of plasma proteins Lipoproteins Hb derivatives Immunoelectrophoresis Agar gel slab Separation of antigenic proteins
- 21. TYPES OF PLASMAPROTEINS Albumin Globulins- α1, α2, β,γ globulins
- 23. How To writein journal Defination Factors affecting electrophoresis Types of electrophoresis Procedure Applications