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Immunogenetics: Open Access

Immunogenetics: Open Access
Open Access


In this technique proteins are separated by two different physicochemical properties. In the first dimension proteins or polypeptides are separated on the basis of their net charges by isoelectric focusing and in the second dimension they are separated on the basis of their molecular masses by electrophoresis. Because it is unlikely that two molecules will be similar in both properties, molecules are more effectively separated in 2-D electrophoresis than in 1-D electrophoresis. The goal of sample preparation is to solubilize maximum number of proteins and maintain their solubility throughout the process. The materials for sample should be carefully collected, snap frozen and ground under liquid nitrogen in the presence of protease inhibitors. After extracting proteins from source material they are then solubilized and denatured by means of chaotropes, detergents, and reducing agents. Hydrogen-bonds in the sample proteins are disrupted by chaotropes urea and thiourea. Uncharged detergents are used to disrupt hydrophobic interactions. Detergents such as CHAPS, Triton X-100, sulfobetaine SB3-10, and amidosulfobetaine are IEF-compatible additives. Disulfide bonds are reduced to sulfhydryls by reducing agents dithiothrietol (DTT), dithioerythritol, and tributyl phosphine (TBP).

Conference Proceedings

Relevant Topics in Immunology & Microbiology