Two-dimensional gel electrophoresis is a high-resolution protein separation method first introduced by O'Farrell and Klose in 1975. This method involves protein separation by two dimensions, mass and charge, making it more accurate than one-dimensional gel electrophoresis.

The first dimension separation uses the isoelectric focusing or IEF technique performed on immobilized pH gradient (IPG) strips that separate proteins according to their isoelectric points.

Biological samples, such as cells and tissues, are first prepared for IEF by treatment with a buffer containing urea, dithiothreitol, detergents, and ampholytes. Here, urea and detergent solubilize and denature the proteins. Dithiothreitol is a reducing agent that cleaves disulfide bonds. Ampholytes used in a suitable concentration solubilize the protein and maintain pH. The buffer also rehydrates the IPG strip that helps it absorb the proteins before their charge-based separation.

Proteins are amphoteric molecules with positive and negative charges. The amphoteric protein molecules migrate on the pH gradient of the IPG strip under the electric current. Once the proteins reach their isoelectric point, they immobilize because, at this point, they have no net charge. IPG strips have a fixed pH gradient; for instance, strips with a smaller pH range (for example, pH 4-7) can detect proteins with higher accuracy than those with a broader range (pH 3-10). Once the proteins separate, the IPG strips are treated with sodium dodecyl sulfate (SDS) buffer to proceed to the next phase, i.e., mass-based separation using SDS-PAGE (polyacrylamide gel electrophoresis). SDS covers the uncharged protein on the IPG strips with a negative charge, and glycerol reduces electroendosmosis which helps the transfer of proteins from the first to the second dimension.

Generally, bands on the gel can be visualized upon staining. Sometimes radiolabelled samples are used to obtain an autoradiographic image. Samples capable of producing fluorescent images can also be used.

Two-dimensional gel electrophoresis has gained importance owing to the precision of the outcome results. However, the method is unsuitable for distinguishing highly hydrophobic proteins, proteins with high molecular mass, or those present in very low quantities per cell.