Definition
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Electrophoresis is the most used method to separate and characterise proteins by applying electric current.
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Electrophoretic procedures are rapid and relatively sensitive requiring only microrgamme quantities of proteins.
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Electrophoresis in polyacrylamide gel is very convenient than in any other medium such as paper and starch gel.
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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.
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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.
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Analysis and comparison of proteins in a large number of samples are easily made on polyacrylamide gel slabs.
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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).
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Solutions are normally degassed by evacuation prior to polymerization since oxygen inhibits polymerization.
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The porosity of gel formed depend upon the concentration of bisacrylamide.
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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%.
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A low percentage gel (with large pore size) is used to separate high molecular weight proteins and vice-versa.
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At high concentrations of persulphate and TEMED the rate of polymerisation is also high.
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Among a number of methods commonly used, the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS – PAGE) in slabs is described below.
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This method permits the characterizations of polypeptides and to determine their molecular weight by co-electrophoresis using a set of standard proteins.
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Last modified: Monday, 19 March 2012, 6:50 AM