Definition

Definition

  • Electrophoresis is the most used method to separate and characterise proteins by applying electric current.
  • Electrophoretic procedures are rapid and relatively sensitive requiring only microrgamme quantities of proteins.
  • Electrophoresis in polyacrylamide gel is very convenient than in any other medium such as paper and starch gel.
  • Electrophoresis of proteins in polyacrylamide gels is carried out in buffer gels (not denaturing) as well as in sodium dodecyl sulphate (SDS) containing (denaturing) gels.
  • Separation in buffer gels relies on both the charge and size of the protein whereas it depends only upon the size in the SDS gels.
  • Analysis and comparison of proteins in a large number of samples are easily made on polyacrylamide gel slabs.
  • Acrylamide gels are formed by polymerising acrylamide with a crosslinking agent (bisacrylamide) in the presence of a catalyst (Persulphate) and chain initiator (TEMED: N,N,N,N – tetramethylene diamine).
  • Solutions are normally degassed by evacuation prior to polymerization since oxygen inhibits polymerization.
  • The porosity of gel formed depend upon the concentration of bisacrylamide.
  •  Gels are usually referred to interms of the total percentage of acrylamide present and most protein separations are performed using gels in the range 7-15%.
  • A low percentage gel (with large pore size) is used to separate high molecular weight proteins and vice-versa.
  •  At high concentrations of persulphate and TEMED the rate of polymerisation is also high.
  • Among a number of methods commonly used, the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS – PAGE) in slabs is described below.
  • This method permits the characterizations of polypeptides and to determine their molecular weight by co-electrophoresis using a set of standard proteins.
Last modified: Monday, 19 March 2012, 6:50 AM