Sodium Dodecyl Sulfate- Polyacrylamide Electrophoresis

• SDS-PAGE - Separates protein molecules according to size, disrupts the secondary, tertiary and quaternary structure of the protein to produce a linear polypeptide chain coated with

  negatively charged SDS molecules
• Mercaptoethanol - assists the protein denaturation by

  reducing all disulfide bonds.
• SDS

  • Anionic detergent
  • When dissolved, it gives a net negative charge
  • Breaks the non-covalent bonds
  • Binds to the amimo acids side chains giving net negative charge
• B-Mercaptoethanol

  • Reducing agent
  • Reduces the disulfide bonds in the proteins
• Tris-HCl PH 6.8 - to provide appropriate pH
• SDS - detergent nature to denature proteins and give them negative charge
• Glycerol - to make samples sink into wells
• Blue dye - to visualize samples as gel is run
• Applications of SDS-PAGE

  • Molecular weight determination
  • Protein profile
  • Protein expression level
  • Purity check
  • Western blot