Chemistry

Protein separation

Protein separation


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Determination of the molecular mass of a protein in a polyacrylamide gel

SDS gel electrophoresis is certainly the most frequently used method for determining the molecular mass of denatured proteins in the laboratory. This method is simple, fast and cheap, but can only be used to a very limited extent for determining the molecular mass of native proteins.

Determination of the molecular weight of unknown proteins in the polyacrylamide gel

In order to determine the apparent molecular mass of an unknown protein in the polyacrylamide gel, a calibration curve is created using the protein standard. The logarithm of the molecular mass of a protein is directly proportional to the migration distance in the gel. In practice, the distance between the band and the top of the gel is determined. The respective values ​​are entered in a diagram for each protein of the standard and result in a straight to slightly sigmoid curve. The molecular mass of each protein from the total bacterial extract can thus be calculated from the calibration curve. A typical protein standard, as it was also used here for the polyacrylamide gel, has, for example, the following composition (M.r = molecular mass)

  • Myosin, M.r 205.000
  • β-galactosidase, M.r 116.000
  • Bovine serum albumin (BSA), M.r 85.000
  • Ovalbumin, M.r 47.000
  • Carbonic anhydrase, M.r 36.800

A calibration curve for the standard can now be created from the molecular mass of a protein and the migration distance in the gel, from which the molecular mass of any protein can be read off.



Comments:

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  4. Negm

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