Electrophoresis basic principle and types.pptx

Electrophoresis basic principle and types

1. Electrophoresis

2. Electrophoresis is a technique used to separate different molecules, like DNA, RNA, or proteins , based on their size and charge. It works by applying an electric current to a gel or solution containing the molecules. Since these molecules have a charge (either positive or negative), they move towards the opposite charge in the electric field. For example: DNA has a negative charge, so it moves towards the positive end Smaller molecules move faster and farther through the gel, while larger ones move slower and stay closer to the starting point. CHARGED MOLECULES - ELECTRIC FIELD- SIZE, SHAPE, AND CHARGE

3. Ferdinand Reuss (1807) : Discovered the principle of electrophoresis. Arne Tiselius (1930) : Pioneered electrophoresis for biological systems, especially protein separation. Oliver Smithies (1950) : Developed gel electrophoresis, improving molecular separation methods. Searching Something New RESEARCH HOW? Why? Why Not?

4. Charged Molecules : Molecules like DNA, RNA, or proteins carry an electrical charge. (Amino Acid, pH) Electric Field : Positively charged molecules move towards the negative electrode (cathode). Medium for Separation (Gel or Solution) : The molecules are placed in a gel (like agarose or polyacrylamide) The gel acts like a "molecular sieve," meaning smaller molecules can move through the gel more quickly, while larger molecules move more slowly. Separation by Size and Charge : 1. Size : 2. Charge : Molecules with a stronger charge move faster towards the electrode, while weaker-charged molecules move slower. Visualization : After electrophoresis, the separated molecules can be visualized using stains or dyes. For DNA, stains like ethidium bromide or SYBR Green dyes are used, and under UV light, the DNA bands can be seen.

5. Rate of migration depends on: 1. Net electrical charge of the molecule 2. Size and shape of the molecule 3. Electrical field strength 4. Properties of the supporting medium 5. Temperature and pH of operation

6. Paper electrophoresis Serum electrophoreisis Starch gel Disc gel Agarose Gel Electrophoresis Polyacrylamide Gel Electrophoresis (PAGE) SDS-PAGE (Sodium Dodecyl Sulfate -PAGE) Isoelectric Focusing (IEF) 2D Gel Electrophoresis (Two-Dimensional Electrophoresis) Pulse-Field Gel Electrophoresis (PFGE) Capillary Zone Electrophoresis Immunoelectrophoresis Agarose Gel Electrophoresis for Proteins Microchip Electrophoresis Disc Electrophoresis Zymography Agarose Gel Electrophoresis of Lipoproteins Starch Gel Electrophoresis Free-Flow Electrophoresis (FFE) Gel Electrophoresis for RNA Denaturing Gradient Gel Electrophoresis (DGGE) Gel Shift Assay Electrophoretic Transfer

7. Buffers: Function include: pH, Electricity The ionic strength influences : Holding capacity Features of an ideal buffer : analytes disturbance X Commonly used buffers include: Low pH (acidic): phosphate, acetate, formate , citrate High pH (basic): tris , tricine , borate, CAPS (N-cyclohexyl-3-aminopropanesulfonic acid) They are good culture media for microorganisms and should be refrigerated when not in use, Also cold buffer reduces evaporation and improves resolution Small volumes should be discarded after use, but large volumes can be reused up to 4 times

8. Commonly used buffers include: Low pH (acidic): phosphate, acetate, formate , citrate High pH (basic): tris , tricine , borate, CAPS (N-cyclohexyl-3-aminopropanesulfonic acid ) Higher the ionic strength (and concentration) > higher size of ionic cloud > lower mobility of the particle Also, higher ionic strength > sharper protein-band separation and increased heat production > denaturation of heat-labile proteins Buffers used are made of monovalent ions because their valencies (ionic strength) and molality are equal. They are good culture media for microorganisms and should be refrigerated when not in use, Also cold buffer reduces evaporation and improves resolution Small volumes should be discarded after use, but large volumes can be reused up to 4 times

9. Support Media: Insoluble gels e.g sheets, slabs or columns of starch, agarose or polyacrylamide Membranes (paper) of cellulose acetate Starch Gel: Separate macromolecules on basis of surface charge and molecular size Obsolete because preparation of reproducible starch gel is difficult Cellulose Acetate: made by treating cellulose with acetic anhydride

10. Agarose: Agarose is a polysaccharide extracted from seaweed that is commonly used in molecular biology to separate DNA, RNA, and proteins based on size. It forms a porous gel when heated and cooled, creating a molecular sieve that allows smaller molecules to pass through more quickly than larger ones. Advantages : Permits excellent densitometry – lower affinity for proteins (migration is not affected) and native clarity after drying Little Endosmosis – free of ionizable groups (neutral) Disadvantage: DNA recovery is affected by inhibitors

11. Polyacrylamide: thermostable, transparent, durable and relatively chemically inert No endosmosis – uncharged Pore size does not allow larger proteins like fibrinogen, B1-lipoprotiens, Y-globulins etc , to migrate Separation is based on both charge-to-mass ratio and molecular mass – molecular sieving Carcinogenic – CAUTION when handling Accommodates a large amount of sample in a single sample slot DNA recovered is pure with no inhibitors unlike Agarose. Separates proteins into 20 or more fractions Used to study individual proteins ( e.g Isoenzymes )

12. Equipment used for Gel Electrophoresis:

13. 1. PAPER ELECTROPHORESIS

14. P aper electrophoresis is simple and inexpensive and requires only micro quantities of sample for separation. The support medium is a filter paper . The electrophoresis apparatus in its simplest form consists of two troughs to contain buffer solution. The charge carried  by a molecule depends on the pH of the medium. Electrophoresis at low voltage is not usually to separate low molecular weight compounds because of diffusion, but it is easier to illustrate the relationship between charge and pH with amino acids than with proteins (or) other macromolecules. The equipment required  for electrophoresis consists basically of two items, a POWER PACK and an ELECTROPHORETIC CELL. Power pack : provides a stabilized direct current and has controls for both voltage & current output, which have an output of 0 to 500V and 0 to 150mA are available.

15. Muscle proteins, egg white proteins, milk proteins & snake, insect venom analysis done by this technique.