Electrophoresis

Electrophoresis lecture

1. TO STUDY ELECTROPHORESIS BY: Kamlesh Yadav ( Msc.Medical Biochemistry)

2. DEFINITION Movement of charged particles (ions) in an electric field resulting in their migration towards the oppositely charged electrode is called electrophoresis. It is widely used analytical technique for the separation of biological molecules such as plasma proteins, lipoproteins.

3. PRINCIPLE Charged molecules migrate either to cathode or anode depending upon the kind of charge they carry. Molecules with a net positive charge ( cations ) move towards cathode whereas molecules with net negative charge (anions) migrate towards anode.

4. FACTORS AFFECTING THE RATE OF MIGRATION Total net charge on molecules. Size and shape of particles. pH of buffer solution. Strength of electric field applied. Property of supporting medium. Temperature

5. REQUIREMENTS FOR ELECTROPHORESIS Electrophoresis tank (to hold buffer). Electrodes Power pack to supply electricity at constant current & voltage. Power supply vary based on type of electrophoresis and type of gel used. (PAGE usually uses higher voltage while Agarose gel electrophoresis generally uses lower voltage).

6. Continued... Buffer To create pH mostly around 8.6. At this pH all serum proteins will have a net negative charge & will migrate towards anode to conduct the current. Specimen (Serum, Plasma, Urine or Fluid CSF, Pleural fluid).

7. TYPES OF ELECTROPHORESIS The most commonly employed electrophoretic techniques in lab include the following. Zone electrophoresis (Paper & Gel) Isoelectric focussing Immuno electrophoresis

8. MOVING BOUNDARY ELECTROPHORESIS Originally developed moving boundary electrophoresis by Tiselius (1937) is less frequently used these days. In this technique U- shaped tube is filled with protein solution overlaid by a buffer solution. As the proteins move in solution during electrophoresis, they form boundaries which can be identified by their refraction index.

9. ZONE ELECTROPHORESIS A simple & modified method of moving boundary electrophoresis. An inert supporting medium such as paper or gel are used. Paper Electrophoresis Gel Electrophoresis

10. PAPER ELECTROPHORESIS Sample is applied on a strip of filter paper wetted with desired buffer solution. The ends of the strip are dipped into buffer reservoirs in which electrodes are placed. The electric currents is applied allowing molecules to migrate for sufficient time. The paper called electrophoretogram is removed dried & stained with a dye that specifically colours the substance to be detected. Serum proteins are separated into separate distinct bands of albumin, alpha 1 globulin, alpha 2 globulins, β-globulins & γ-globulins.

11. GEL ELECTROPHORESIS Electrophoresis that involves the use of a gelatinous material as agarose , acrylamide , starch or cellulose acetate as the matrix (support medium). It involves separation of molecules based on their size in addition to electrical charge. Movement of large molecules is slow. Resolution is much higher & thus, serum proteins can be separated to about twenty different bands instead of five bands on paper electrophoresis. Polyacrylamide gel electophoresis (PAGE) is one of the commonly used technique named after the gel used.

12. POLYACRYLAMIDE GEL ELECTROPHOREIS Electrophoresis is carried out in polyacrylamide gel with a characteristic pore size. Proteins are separated on the basis of their charge and molecular size. In this technique, serum sample is applied at the top of gel, and proteins, following electrophoresis, are stained with amido black. It may yield 20 or more fractions. widely used to study individual proteins in serum, genetic variants and isoenzymes .

13. PAGE can also be carried out in the presence of sodiumdodecyl sulphate (SDS), called as SDS-PAGE.

14. SDS-PAGE Stands for sodiumdodecyl sulphate- polyacrylamide gel electrophoresis. Variant of PAGE. Proteins are boiled for one or two minutes with SDS which is a denaturing agent. The negative charges of SDS cover the protein molecules making them strong negative. Then the separation will depend mainly on molecular size. It is commonly used for molecules weight determination as well as for assessing the purity of proteins.

15. ISOELECTRIC FOCUSSING Primary based on immobilization of molecule at iso -electric pH during electrophoresis. Serum proteins can be separated to as many as 40 bands. Can be conveniently used for the purification of proteins.

16. IMMUNO ELECTROPHORESIS It involves combination of the principles of electrophoresis & immunological reactions. Used for analysis of complex mixture of antigens & antibodies.

17. APPLICATIONS DNA sequencing Blotting Medical Research Protein Research Drugs Vitamins Carbohydrates Agricultural testings

18. CLINICAL APPLICATIONS DISEASE Hepatic Cirrhosis Nephrotic Syndrome Multiple myeloma Protein energy malnutrition Acute infection Chronic infection ELECTROPHORESIS FINDINGS Albumin decrease/ band γ-Globulin increases. Alpha-2-globulin increases N-band seen between β & γ globulin. Albumin decreases Increase in α-2-globulin. Increase in both α-1 & α-2 globulin.

19. Thank you