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Principle

Isoelectric focusing gel electrophoresis is a separation of amphoteric substance such as protein, amino acids and peptides based on their different isoelectric point on supporting medium using electricity.

Gel

Instead of using buffer of constant pH, a pH gradient is maintained along the gel.
The pH gradient is maintained by using polyvalent synthetic ampholytes.
The ampholytes can be purchased in either wide band pH range (pH 3 to 10) or narrow range (pH 7 to 8).
A pH range is chosen such that the sample being separated will have their Isoelectric point within this range in Isoelectric focusing gel electrophoresis.
E.g. commercially available ampholytes are biocyte, pharmalyte, ampholine

Sample preparation and application

In Isoelectric focusing gel electrophoresis the sample is mixed with suitable buffer and applied on the gel using square filter paper bits soaked in the sample solution. A voltage is applied for about 30mm for the sample to diffuse from the paper into gel. The filter paper bits are then removed.

Sample separation

Once the sample is loaded in isoelectric focusing gel electrophoresis a voltage is applied. If the protein sample to be separated is loaded initially below the isoelectric point, they will be positively charged and will initially migrate towards cathode and anode. The pH of the surrounding steadily increase and therefore the positive charge on the protein will decrease eventually and the protein arrives at the point where the pH is equal to its isoelectric point. The protein will now be in the zwitter ion form with no net charge.

Staining and Destaining

Following Isoelectric focusing gel electrophoresis the gels must be stained to detect the protein. But this cannot be done directly because the ampholytes will stain giving a totally blue gel. The gel is therefore first washed with fixing solution 10% TCA. This precipitates the protein in the gel. The gel is now stained with CBB and then destained.