2DGelelektrophorese

Two-dimensional gel electrophoresis (2DGE) is a proteomics technique that resolves complex protein mixtures by separating proteins in two orthogonal dimensions: first by isoelectric point (pI) using isoelectric focusing on immobilized pH gradient strips, and then by molecular weight using SDS-PAGE. In German, the method is commonly referred to as 2D-Gelelektrophorese.

Preparation begins with solubilizing proteins in chaotropic agents and detergents (for example urea, thiourea, CHAPS) to unfold them. The solubilized sample is loaded onto immobilized pH gradient strips for isoelectric focusing, where proteins migrate to the pH corresponding to their pI and focus into sharp bands. After focusing, the strips are equilibrated in SDS-containing buffer to impart a uniform negative charge and to reduce disulfide bonds, enabling the second-dimension separation. The strips are then laid on or inserted atop a denaturing polyacrylamide gel, and the proteins are separated by molecular weight during electrophoresis.

Visualization and analysis are performed by staining the gel (for example Coomassie, silver, or fluorescent dyes) to produce a two-dimensional map of protein spots. Image analysis software is used to align spots across samples, quantify spot intensities, and select candidates for identification by in-gel digestion followed by mass spectrometry.

Variants such as 2D-DIGE (differential in-gel electrophoresis) label different samples with distinct fluorescent dyes and are run on the same gel to improve quantitative accuracy. Limitations include relatively low throughput, labor intensity, substantial sample preparation requirements, bias against very hydrophobic or extreme-molecular-weight proteins, and limited dynamic range compared with modern shotgun proteomics.