Two Dimensional PAGE for Proteome Analysis

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During last lectures we have studied electrophoretic methods of separation of protein based on mass (PAGE) or isoelectric point (Isoelectric focusing). However, simplest bacteria may have over 4000 individual proteins while eukaryotes the number may be approximately 50,000.

  In the large population of protein it may be possible that more than one protein has identical (or close) molecular mass and may not be separated by PAGE. It is also possible that more than one protein has very close isoelectric point (pI) and separation is not possible by isoelectric focusing (IEF) . Combination of these two methods may yield better separation.

  Two-dimensional gel electrophoresis (2-D electrophoresis) is widely used technique for the analysis of complex protein mixtures. As explained above, this method separate proteins in two steps, the first-dimension is isoelectric focusing, which separates proteins according to their isoelectric points (as we have studied in our previous lectures); the second-dimension is SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which separates proteins according to their molecular weights (as we have studied in our previous lectures). In this way, complex mixtures consisted of thousands of different proteins can be separated.