Gel electrophoresis

materials and method

1. GEL ELECTROPHORESIS Chethan. N 1 st M Pharm Dept. of Pharmacology, J.S.S.C.P, MYSORE .

2. Positive or negative electrical charges are frequently associated with biomolecules. When placed in an electric field, charged biomolecules move towards the electrode of opposite charge due to the phenomenon of electrostatic attraction Introduction :

3. Electrophoresis Electrophoresis is the separation of charged molecules in an applied electric field. The relative mobility of individual molecule depends on several factors. The most important of which are: Net charge Charge/mass ratio, Molecular shape and The temperature, porosity and viscosity of the matrix through which the molecule migrates.

4. Gel Electrophoresis Gel electrophoresis is a method for separation and analysis of macromolecules like DNA, RNA and proteins or their fragments, based on their size and charge. Gel electrophoresis uses a gel as an anti-convective medium and/or sieving medium during electrophoresis. Gels suppress the thermal convection caused by application of the electric field, gels can also simply serve to maintain the finished separation, so that a post electrophoresis stain can be applied.

5. Principle By placing the substance to be separated in wells of the gel and applying an electric current, allows the molecule to move through the matrix at different rates towards the anode if negatively charged or toward the cathode if positively charged. As they move through the gel, the larger molecules will be held up as they try to pass through the pores of the gel, while the smaller molecules will be impeded less and move faster. This results in a separation by size, with the larger molecules nearer the well and the smaller molecules farther away.

6. Principle of separation According to charge: When charged molecules are placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge. According to size: The smaller molecules move more swiftly than the larger sized ones, as the can travel through the pores more easily than the later.

7. Instrument and reagents Electrophoresis apparatus Buffer Power supply Supporting media Detection and Quantification

8. 1.Electrophoresis apparatus: The casting tray is made up of glass or plastic. The comb contains varying number of teeth in order to help in formation of well.

9. Electrophoresis apparatus set up: Electrophoresis chamber with buffer solution Casting tray Electrodes

10. 2. Buffer: Buffers in gel electrophoresis are used to provide ions that carry a current and to maintain the pH at a relatively constant value. The most common being, for nucleic acids Tris/Acetate/EDTA (TAE), Tris/Borate/EDTA(TBE).

11. 3. Power supply: The electrodes are connected to their respective terminals of the electrophoresis chamber and to the power supplier with controls for rate of current flow. The best resolution of fragments larger than about 2 kb is attained by applying no more than 5 volts per cm to the gel

12. 4. Supporting media: (Gel) Starch Agar/agarose Cellulose acetate Polyacrylamide gel The kind of supporting matrix used depends on type of molecules to be separated and the desired basis for separation: charge, molecular weight or both

13. Agarose and polyacrylamide gels are cross-linked, spongelike structure It is important that the support media is electrically neutral. Presence of charge group may cause: -Migration retardation -The flow of water toward one or the other electrode so called ‘ Electroendosmosis (EEO) ’ , which decrease resolution of the separation Agarose Gels have fairly large pore sizes and are used for separating larger DNA molecules (Restriction Fragment Length Polymorphism Analysis) Polyacrylamide Gels are used to obtain high resolution separations for smaller DNA molecules (STR analysis and DNA sequence analysis)

14. 5. Detection and quantification: Stains Protein staining Ethidium bromide staining Blotting Southern blotting (for DNA) Northern blotting (for RNA) Western blotting (for protein)

15. Ethidium bromide Protein staining

16. Process of gel electrophoresis

17. Gel electrophoresis Agarose gel - electrode + electrode DNA fragments buffer ~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~ - electrode + electrode current buffer ~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~

18. visualization The molecules in the gel are stained to make them visible. DNA may be visualized using ethidium bromide which, when intercalated into DNA, fluoresce under ultraviolet light, while protein may be visualised using silver stain or Coomassie Brilliant Blue dye. SYBR Green I is more expensive, but 25 times more sensitive and possible safer than ethidium bromide. SYBR Safe is a variant of SYBR Green, and show low level of mutagenicity and toxicity. Other less frequently used markers are Cresol red and Orange G.

19. Factors affecting separation in gel electrophoresis The sample: The charge/mass ratio of the sample dictates its electrophoretic mobility. Charge: Higher the charge, greater the electrophoretic mobility. Size: Size is inversely proportional to electrophoretic mobility. Shape: Globular substances move faster than the fibrous ones.

20. The electric field: An increase in the potential gradient increases the rate of migration. The medium: T he inert medium can exert adsorption or molecular sieving effects on the particle influencng its rate of migration. Adsorption: retention of the component on the surface of supporting medium. Molecular sieving: media such as agar, polyacrylamide, sephadex have cross linked structures giving rise to pores within the gel beads.

21. The buffer: the buffer can affect the electrophoretic mobility by: Ionic strength: increase in ionic strength of buffer means a larger share of current is carried by buffer and smaller proportion by sample, while decrease in ionic strength is vice-versa. pH: pH determines the degree of ionization of organic compounds. Where ionization is inversely proportional to pH.

22. Applications Separation of Deoxyribonucleic acid Separation of ribonucleic acid Separation of protein molecules It may be used as preparative technique prior to use of other methods such as mass spectroscopy, cloning, DNA Sequences, Southern Blotting for further characterization. Separation of amino acid Separation of lipoproteins Separation of enzyme in blood Separation of antibiotic drug    

23. References : Instrumental methods of chemical analysis. By Dr. B.K.Sharma, Page no. 661-670 . Instrumental analysis by William Kemp. http://www.intechopen.com/books/gel-electrophoresisprinciples-and-basics Wikipedia .

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