ELECTROPHORESIS

ELECTROPHORESIS

1. Electrophoresis Gadham . Rajeev Email:[email protected]

2. The movement of charged particles (ions) in an electric field resulting in their migration towards the oppositely charged electrode is known as electrophoresis. Used for the separation of plasma proteins, lipoproteins & immunoglobulins . Electrophoresis

3. Electrophoresis

4. The rate of migration of ions in an electric field depends on several factors that include shape, size, net charge and solvation of the ions, viscosity of the solution & magnitude of the current employed

5. In this technique, the sample is applied on a strip of filter paper wetted with desired buffer solution. The ends of the strip are dipped into the buffer reservoirs in which the electrodes are placed . The electric current is applied allowing the molecules to migrate for sufficient time . Paper electrophoresis

6. Paper is removed, dried & stained with dye. The colored spots can be identified by comparing with a set of standards run simultaneously. For serum proteins, whatman No.1 filter paper, tris buffer pH8.6 & stains amido black bromophenol blue are used. Serum proteins are separated into five bands- albumin , α 1, α 2, β & γ - globulins.

7. It involves the separation of molecules based on their size, in addition to the electrical charge. The large molecules move slowly in this. The resolution is much higher in this technique. Serum proteins can be separated to about 15 bands . Gel electrophoresis

8. Commonly used gel are agarose & polyacrylamide, sodium dodecyl sulfate (SDS). Polyacrylamide is employed for the determination of molecular weights of proteins – known as SDS-PAGE .

9. It is based on the immobilization of the molecules at isoelectric pH. Stable pH gradients are set up covering the pH range to include the isoelectric points of the components in a mixture. As the electrophoresis occurs, the molecules (say proteins) migrate to positions corresponding to their isoelectric points, get immobilized & form sharp stationary bonds. Isoelectric focussing

10. The gel blocks can be stained & identified. By isoelectric focussing , serum proteins can be separated to as many as 40 bands. lsoelectric focussing can be conveniently used for the purification of proteins.

11. This technique involves combination of the principles of electrophoresis & immunological reactions. lmmunoelectrophoresis is useful for the analysis of complex mixtures of antigens & antibodies. Immunoelectrophoresis

12. Textbook of Biochemistry – U S atyanarayana References

13. Thank You