2D gel electrophoresis

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It is a powerful method of separating protein along a tubular gel using iso-electric focusing and sodium dodecyl sulphate polyacrylamide gel. The sample undergoes iso-electric focusing first; an electrical current is passed through a gel with a pH gradient, and the proteins stop moving when they reach the pH at which they have no net charge - this is known as the Iso-electric point (pI). The sample is then placed horizontally on top of SDS-PAGE and spreads across. The sample then moves vertically down again to yield the second dimension based on the size of the protein, producing a 2D pattern. So the sample has been separated by virtue of Iso-electric point (pI)[1], when they move horizontally and by virtue of their size; when they move down vertically. 

Because of the specificity of this method, it is possible to identify over one thousand proteins on the same gel, in one experiment [2].

The concentration of isolated proteins can be determined by measuring the intensity of the spots on the gel. Comparing this to other cells can show differences due to differing physiological conditions [3].

This method can be used to separate DNA fragments because the phosphate backbone of DNA has a negative charge. The distance a DNA fragment travels is inversely proportional to the log of its molecular weight.

Reference

  1. Berg , j. Toya, W. 1995. biochemistry, 7 edition, new york
  2. Stryer, Lubert; Tymoczko, John L.; Berg, Jeremy M., (2012) Biochemistry, 7th Edition, New York: WH Freeman and Co. Page 74
  3. Two- dimensional electrophoresis: (Page 76) Berg J, Tymoczko J, Stryer L. Biochemistry. Seventh Edition. New York: W.H. Freeman; 2012.
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