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Gel electrophoresis himanshu

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Introduction
Gel Electrophoresis
Principle of separation
Instrument and reagents
Factors affecting separation in gel electrophoresis
Applications
Electrophoresis apparatus
Buffer
Power supply
Supporting media
Detection and Quantification
Agarose
Polyacrylamide

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Gel electrophoresis himanshu

  1. 1. Gel Electrophoresis Himanshu Kamboj Assistant Professor
  2. 2. ⚫Introduction ⚫Gel Electrophoresis ⚫Principle of separation ⚫Instrument and reagents ⚫Factors affecting separation in gel electrophoresis ⚫Applications Contents
  3. 3. • 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
  4. 4. 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:  Netcharge  Charge/massratio,  Molecularshape and  The temperature, porosity and viscosity of the matrix through which the molecule migrates.
  5. 5. 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 postelectrophoresisstain can be applied.
  6. 6. • Gel material acts as a "molecular sieve”. • Gel is a colloid in a solid form (99% is water). • It is important that the support media is electrically neutral. • Different types of gels which can be used are; Agar and Agarose gel, Starch, Sephadex, Polyacrylamide gels.
  7. 7. 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.
  8. 8. 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.
  9. 9. Instrument and reagents 1. Electrophoresis apparatus 2. Buffer 3. Power supply 4. Supporting media 5. Detection and Quantification
  10. 10. 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.
  11. 11. Electrophoresis apparatus set up: •Electrophoresis chamberwith buffer solution •Casting tray •Electrodes
  12. 12. 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).
  13. 13. 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
  14. 14. 4. Supporting media: (Gel) 1. Starch 2. Agar/agarose 3. Cellulose acetate 4. 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
  15. 15. ⚫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)
  16. 16. Agarose  Polysaccharide extracted from sea weed.  Gel casted horizontally  Non-toxic.  Separate large molecules  Commonly used for DNA separations.  Staining can be done before or pouring the gel. Polyacrylamide gel  Cross-linked polymer of acrylamide.  Gel casted vertically.  Potent neuro-toxic.  Separate small molecules.  Used for DNA or protein separations.  Staining can be done after pouring the gel. Agarose Polyacrylamide Gel
  17. 17. 5. Detection and quantification: ⚫Stains ⚫Protein staining ⚫Ethidium bromide staining ⚫Blotting ⚫Southern blotting (for DNA) ⚫Northern blotting (for RNA) ⚫Western blotting (for protein)
  18. 18. Ethidium bromide Protein staining
  19. 19. Process of gel electrophoresis
  20. 20. Gel electrophoresis - electrode + electrode DNA fragments Agarose gel ~~~~~~~~~~~~~~~~~~~~~~~~ buffer ~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~ - electrode + electrode current ~~~~~~~~~~~~~~~~~~~~~~~~ buffer ~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~~~~~~~
  21. 21. 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.
  22. 22. Gel electrophoresis 1. Agarose gel electrophoresis 2. Starch gel electrophoresis 3. Polyacrylamide gel electrophoresis
  23. 23. Agarose gel electrophoresis  Commonly used support medium  Less expensive than cellulose acetate  Equally good separation  Agar is a complex acidic polysaccharide containing monomers of sulfated galactose  Agarose is a sulfate free fraction of Agar  Gel is prepared in buffer and spread over a microscopic slide  A small sample of serum or biological fluid is applied by cutting in to the gel with a sharp edge  The electrophoretic rum takes about 90 minutes
  24. 24. ADVANTAGES  Easy to prepare and small concentration of agar is required.  Resolution is superior to that of filter paper.  Large quantities of proteins can be separated and recovered.  Adsorption of negatively charged protein molecule is  negligible.  It adsorbs proteins relatively less when compared to other  medium.  Sharp zones are obtained due to less adsorption.  Recovery of protein is good, good method for preparative  purpose.
  25. 25. Factors affecting separation in gel electrophoresis ⚫Thesample: ⚫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 fibrousones.
  26. 26. ⚫The electric field: An increase in the potential gradient increases the rateof migration. ⚫The medium: The inert medium can exert adsorption or molecular sieving effects on the particle influencng its rateof migration. ⚫Adsorption: retention of the component on the surfaceof supporting medium. ⚫Molecular sieving: media such as polyacrylamide, sephadex have cross agar, linked structures giving rise to pores within the gel beads.
  27. 27. ⚫The buffer: the buffercan affect theelectrophoretic mobility by: ⚫Ionic strength: increase in ionic strength of buffer means a larger share of current is carried by bufferand smaller proportion by sample, while decrease in ionic strength isvice-versa. ⚫pH: pH determines the degree of ionization of organic compounds. Where ionization is inversely proportional to pH.
  28. 28. 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
  29. 29. THANK YOU

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