Principle of 2-D Electrophoresis

The sample is denatured with a lysis buffer, the first dimension is isoelectric focusing in presence of 8 or 9 molar urea and a non-ionic detergent. SDS electrophoresis is run as the second dimension.

The sample mixtures are first separated in narrow film-supported gel strips containing fixed buffering groups, which form a pH gradient. The strips are cut from a dried 0.5 mm thin slab gel containing this immobilized pH gradient and rehydrated in the sample solution.

With the application of the electric field the proteins migrate towards their isoelectric points and become focused. They are separated according their isoelectric points.

Prior to SDS-PAGE the strips have to be equilibrated in SDS buffer. The proteins become unfolded and negatively charged because of the bound detergent SDS.

The IPG strip is placed on the SDS gel edge to edge. In the second dimension the proteins are separated according their molecular weights.