Electrophoresis

INTRODUCTION, DEFINATION OF ELECTROPHORESIS, ELECTROPHORESIS PRINCIPLE, TYPES OF ELECTROPHORESIS, FREE ELECTROPHORESIS, ZONE ELECTROPHORESIS,PAPER ELECTROPHORESIS, WORKING OF PAPER ELECTROPHORESIS, PROCEDURE FOR PAPER ELECTROPHORESIS, VISUALISATION, FACTORS AFFECTING SEPARATION OF MOLECULES, APPLICATIONS, working of paper electrophoresis ,procedure for paper electrophoresis ,visualisation ,factors affecting separation of molecules ,applications ,forensics ,dna fingerprinting ,molecular biology ,microbiology information about the organisms ,biochemistry mapping of cellular components ,paper electrophoresis is also used in study of sic ,hemoglobin abnormalities ,separation of blood clotting factors ,serum plasma proteins from blood sample ,used in separation and identification of alkaloids ,used for testing water samples ,toxicity of water ,drug industry to determine presence of illelgal drUGS

1. Electrophoresis Prepared By: Vipin Kr Shukla Assistant Lecturer

2. Introduction: 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. Depending on kind of charge the molecule carry, they move towards either: To cathode Or to Anode An ampholyte become positively charged in acidic condition and migrate to cathode, in alkaline condition they become negatively charge and migrate to anode.

3. Electrophoresis Apparatus:

4. Contd…. It is a type of protein separation method which relies on protein sizes to segregate the mixture. It is one of the highly efficient techniques of analysis and sole method for separation of proteins for western blot, RNA studies etc. But, on negative side it also time-consuming, expensive and technical skilled procedure due to which is less preferred in health care Electrophoresis is similar to other separation techniques like chromatography but it differs in-terms of the types of samples analyzed, the method used for separation, principle used etc.

5. ELECTROPHORESIS & ITS PRINCIPLE: Electrophoresis is a method of separation where in charged molecules migrate in differential speeds in an applied electric field . W hen electricity is applied to the medium containing biological molecules , depending on their net charge and molecular size, they migrate differentially, thus different proteins/DNA can be separated. Depending on the kind of charge the molecule carry, they move towards either to To cathode or anode.

6. FREE ELECTROPHORESIS: In this type of electrophoresis a free electrolyte is taken in place of supporting media. It is mostly of two types : Micro Electrophoresis : It is mostly used in calculating Zeta potentials(a colloidal property of cells in a liquid medium)of the cells. Moving boundary Electrophoresis which for many years had been used for quantitative analysis of complex mixtures of macromolecules , esp. Proteins.

7. ZONE ELECTROPHOROSIS: It involves the migration of the charged particle on the supporting media can be Paper , Cellulose acetate membrane, Starch Gel, Polyacrylamide. Components separated are distributed into discrete zone on the support media. Supporting media is saturated with buffer solution, small volume of the sample is applied as narrow band. ADVANTAGES : Useful in biochemical investigations. Small quantity of sample can be analyzed. Cost is low and easy maintenance. DISADVANTAGES: Unsuitable for accurate mobility and isoelectric point determination. Due to the presence of supporting medium, technical complications.

8. PAPER ELECTROPHORESIS: Paper Electrophoresis is one of the type of zone electrophoresis . Principle: When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge . In contrast to proteins , which can have either a net positive or net negative charge, nucleic acids have a consistent negative charge imparted by their phosphate backbone, and migrate towards the anode.

9. EQUIPMENTS: The equipment required for electrophoresis consist a basically of two items, a POWER PACK and ELECTROPHORETIC CELL . 1. Power pack: Power pack provides a stabilized direct current & has controls for both voltage & current out put, which have an out put of 0 to 500V and 0 to 150mA are available . 2. The Electrophoretic cell: It contains: the electrodes, buffer reservoirs, a support for paper and a transparent insulating cover. The electrodes are usually made of platinum .

10. WORKING: 1 ) A long strip of filter paper is moistened with a suitable buffer solution of the desired p H and the sample is applied transversely across the central part of the strip. 2) Ends are fixed to dip in buffer solutions in two troughs fitted with electrodes. 3)Electric field of about 20 volts/cm is established. 4)The charged particles of sample migrate along the strip towards respective electrodes of opposite polarity, according to net charges, sizes and interactions with the solid matrix.

11. Contd…. 5)Homogeneous group of particles migrate as a separate band 6)The electrophoresis is carried out for 16-18 hours. 7)Proteins are stained (Bromphenol blue) to make them visible 8) The separated proteins appear as distinct bands.

12. PROCEDURE FOR ELECTROPHORESIS

13. This ensures that the electric current goes through the whole tank and that maintains that ions can move in the solution

14. Safety cover is put over the top and the current is switched on. The dye will migrate through the gel toward the positive electrode, as will the DNA. Depending on how much voltage is applied and how warm the gel is and size and shape of molecules will depend on how fast the ions move through the gel. Smaller fragments will move easier so they will be closer to the positive electrode. Once the dye has moved through the gel to the buffer, the electrical current is switched off and gel is removed from the tray.

15. VISUALISATION: After the electrophoresis is complete, the molecules in the gel can be stained to make them visible. Ethidium bromide, silver, or coomassie blue dye may be used for this process. If the analyte molecules fluoresce under ultraviolet light, a photograph can be taken of the gel under ultraviolet lighting conditions. If the molecules to be separated contain radioactivity added for visibility, an autoradiogram can be recorded of the gel .

16. FACTORS AFFECTING SEPARATION: The Sample- Charge- Higher the charge greater the mobility Size- Bigger the molecule greater the frictional and electrostatic forces exerted on it by the medium i.e. larger particles have smaller electrophoretic mobility compared to smaller particles. Shape- The globular protein will migrate faster than the fibrous protein.

17. Electric field: Increase of migration with the increase of voltage gradient. Buffer- Migration of charge particle depend on of the buffer. Composition Commonly used buffers are “Formate”, “Acetate ”, “ Citrate”, “ Phosphate”, “EDTA ”. The choice of buffer depends upon the type of sample being electrophoresed.

18. pH: The extent of ionization depends on pH, especially in organic compounds. The ionization increases with increase in pH of an organic acids and its just reverse for the organic bases therefore affecting its rate of migration .. The Medium : The inert medium can exert adsorption , molecular sieving effects & electro-osmosis – processes that affect the electrophoretic rate. Adsorption : It means retention of a component on the surface of supporting medium. The rate and resolution of the electrophoretic separation can be efficiently reduced by adsorption .

19. APPLICATIONS: Paper electrophoresis has emerged as a simple, inexpensive , and accurate laboratory procedure for various research and clinical studies. Clinical applications of paper electrophoresis include study of sickle cell disease, hemoglobin abnormalities, and separation of blood clotting factors and serum plasma proteins from blood sample .

20. Contd…. It has also been used in separation and identification of alkaloids . PE can also be used for testing water samples, toxicity of water , and other environmental components. Drug-testing industry uses paper electrophoresis to determine presence of illegal drugs crime suspects.

21. Contd…. Forensics. DNA fingerprint of a criminal. Molecular Biology To separate and organize DNA and RNA by size. Genetics Provide clearer picture of DNA, it also helps prepare DNA for cloning and genetic engineering. Microbiology Information out about the organisms. Virology : to help diagnose different strains of viruses. Biochemistry Mapping of cellular components, particularly proteins and nucleic acids .