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Polyacrylamide Gel Electrophoresis (SDS-PAGE & Native PAGE)

1. Polyacrylamide Gel Electrophoresis Techniques and Instrumentation (PAGE) Abhishikt David Solomon PID No: 16BTBIOT001 B.Tech Biotechnology 2. PAGE- Polyacrylamide Gel…
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  • 1. Polyacrylamide Gel Electrophoresis Techniques and Instrumentation (PAGE) Abhishikt David Solomon PID No: 16BTBIOT001 B.Tech Biotechnology
  • 2. PAGE- Polyacrylamide Gel Electrophoresis One of the 3 types of gel elctrophoresis involving the polyacrylamide gel used as a supporting media. Polyacrylamide is produced as a result of the polymerization reaction between acrylamide and N,N'-methylene-bis- acrylamide (BIS) using a catalyst.
  • 3. Polyacrylamide is used to separate fragments less than 500 bp. It is the most widely used technique of gel electrophoresis. PAGE is of 2 types: • SDS- PAGE • Native- PAGE
  • 4. SDS- PAGE • Modified version of PAGE in which SDS- Sodium dodecyl sulfate is used. • Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a very common method of gel electrophoresis for separating proteins by mass. • SDS-PAGE was first known as the Laemmli method, after its inventor, U.K. Laemmli.
  • 5. • SDS is an amphipathic surfectant, i.e, it acts as both hydrophobic and hydrophilic species. • It denatures protein chain and gives protein a net negative charge. • It coats the protein chain with surfectant molecules.
  • 6. Sample: • Tris-HCl : provides necessary Cl ions • SDS: detergent and denaturation of protein structures • - Mercaptoethanol: overcomes tertiary protein structures and reduces sulphide bonds. • Bromophenol Blue: staining dye • Glycerol: increases viscosity of sample • ddH20: to make the sample ion free • • Migration buffer is poured into the gel plate on both the sides of the electrodes which contain Tris-Gly.
  • 7. Gel Preparation: • Tris-HCl • Acrylamide mix • SDS 10% • Ammonium persulfate (gives free radical after decomposing and polymerizes acrylamide and bisacrylamide) • TEMED (catalyst for polyacrylamide gel polymerization)
  • 8. Stacking Gel (4%)- • It has a low pH of 6.8. • It is of low concentration and big pore size. • It is the upper portion of the gel from where the migration of the sample starts.
  • 9. The sample contains the following ions- • chloride ions which migrate at a higher speed in both the gels and hence are called the leading ions , • glycine ions, which are present in the migration buffer (Tris- Gly) and are known as the trailing ions due to their migration at low speeds, and • proteins in between the chloride and glycine ions which are stacked as narrow as possible so that they may enter the resolving gel.
  • 10. Being a negatively charged ion, glycine from the Tris-Gly buffer (pH 8.3) will migrate slowly in the stacking gel region due to low its low pH 6.8 and will be increased in the separating gel due to its high pH 8.8.
  • 11. Resolving or Separating Gel (10%)- • It has a high pH of 8.8. • More concentrated than the stacking gel. • The chloride ion will migrate the fastest in this gel, followed by glycine (due to an increase in pH) and proteins which are separated in this region.
  • 12. NATIVE PAGE Native – original form Separation of proteins in its original or intact form. Separation is on the basis of charge, mass and shape of proteins.
  • 13. • Charges are not separate from the proteins as in the case of SDS-PAGE. • • Proteins are not treated in the denatured conditions. • Enzymes added to the NATIVE- PAGE are not denatured due to which they can be detected by this technique. • Globular proteins migrate faster than the elongated proteins. • Larger the proteins, slower is the migration due to the molecular seive formed in the gel.
  • 14. APPLICATIONS OF PAGE • Detection of Amino acid content. • Protein-protein interaction. • Detection of enzymes. • To analyze RNA samples
  • 15. THANK YOU
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