2DE

2DE stands for two-dimensional gel electrophoresis, a proteomic method used to separate complex mixtures of proteins. It combines two sequential separation steps to achieve high resolution of protein species.

Principle: In the first dimension, proteins are separated by their isoelectric point (pI) using isoelectric focusing on immobilized pH gradient strips. In the second dimension, the proteins are separated by molecular weight using SDS-PAGE. The combined separation disperses thousands of proteins into distinct spots on a two-dimensional gel.

Procedure: Proteins are solubilized in a denaturing buffer and applied to the IPG strip for focusing. After focusing, the strip is equilibrated in reducing and alkylating buffers and then laid onto an SDS-PAGE gel for the second dimension. Following separation, proteins are visualized by staining or fluorescence; spots are detected with imaging systems and analyzed computationally to compare patterns across samples.

Variants and improvements: 2D-DIGE (two-dimensional difference gel electrophoresis) uses fluorescent dyes to label samples for simultaneous analysis in one gel, improving quantitative comparison. Other approaches combine 2DE with targeted or discovery mass spectrometry for protein identification after spot excision.

Strengths and limitations: The technique offers high resolving power and can reveal protein isoforms and certain post-translational modifications. Limitations include labor intensity, long run times, a limited dynamic range, and underrepresentation of very basic or very acidic, highly hydrophobic, or membrane proteins. Reproducibility between gels and accurate spot matching can also be challenging.

Applications: 2DE is used for global proteome profiling, comparative proteomics, biomarker discovery, and isoform analysis, particularly in laboratories pursuing detailed protein separation patterns prior to identification.