2d dige protein identification

Difference Gel Electrophoresis (DIGE) is a technology, that enables separation and quantification of fluorescent-labeled proteins (Cy2, Cy3, and Cy5) from different samples through a two dimensional gel electrophoresis. Protein samples are differentially labeled using three cyanine fluorescent dyes (Cy2, Cy3 and Cy5; Amersham Biosciences) prior to gel electrophoresis. Groups of labeled proteins are then mixed and separated by one 2D-gel. At the first dimension of the 2D-gel, proteins are separated by IEF, which is based on the isoelectric points of proteins. In the second dimension, proteins are separated in PAGE gel based on the molecular weight of proteins. Afterwards, fluorescent images at each channel that are used for detection of corresponding fluorescent-labeled proteins are captured. image from each channel is superimposed with one another, which are automatically analyzed by the DIGE software. Software can identify the significant distinct spot to be sent for mass spectrometry analysis for further quantification of the proteins in the picked spot.