Two-dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis combines two dimensions for protein separation: first based on charge, and second by mass.

The first dimension uses the method of isoelectric focusing or IEF. Here, the protein sample is loaded on an immobilized pH gradient or IPG strip.

On applying electric current, the proteins move across the pH gradient on the strip, immobilizing at their isoelectric point - the pH, where the proteins carry no net charge.

Next, the IPG strip is treated with SDS and loaded onto a polyacrylamide gel for separation by the second dimension using SDS-PAGE.

In a direction perpendicular to IEF, the proteins separate electrophoretically based on mass.

The separated proteins are then visualized post staining.

Two-dimensional gel electrophoresis is a high-resolution technique that can identify similar proteins differing by even one charged amino acid residue.

Further, it can detect protein modifications inside a cell or organelle during different conditions and developmental stages.