2D DIGE Analysis of Protein Extracts from Muscle Tissue - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 155

Chapter 11
2D DIGE Analysis of Protein Extracts from Muscle Tissue
Cecilia Gelfi and Sara De Palma
Abstract
2D DIGE, two-dimensional difference gel electrophoresis, is a technology used to study the protein
expression on two-dimensional gels. Protein samples are labeled with different color fl uorescent dyes
designed not to affect the relative migration of proteins during electrophoresis. Here, we describe the
practical procedures necessary to perform a 2D DIGE experiment for a muscle tissue protein extract fol-
lowed by CyDye DIGE fl uors minimal labeling and the analysis of 2D DIGE gels for the assessment of
quantitative differences.
Key words: 2D DIGE, 2D electrophoresis, CyDye DIGE fl uors, Differential analysis, Muscle
proteome
1. Introduction
The DIGE technique was fi rst described in 1997 ( 1 ); it is a modifi ed
form of 2DE, two-dimensional electrophoresis ( 2-4 ), developed
to overcome the problem of the irreproducibility of gels and to
increase the sensitivity of 2DE methodology by labeling samples
with a different fl uorescent dye prior to running them on the same
gel (see Fig. 1 ).
Protein samples are labeled with up to three spectrally distinct,
charge- and mass-matched fl uorescent dyes (CyDye DIGE fl uors)
( 1, 5, 6 ): Cy2, Cy3, and Cy5. This means that the same protein
labeled with any of the CyDye DIGE fl uors migrates to the same
position on a 2D gel ( 7 ) eliminating intragel variation. Moreover,
the dyes afford great sensitivity with a detection capability of 125 pg
of a single protein and a linear response over at least fi ve orders of
magnitude. CyDyes DIGE fl uors have an N -hydroxysuccinimide
ester reactive group and are designed to covalently attach to the
epsilon amino group of lysine via an amine linkage (see Fig. 2 ). The
quantity of dye added to the sample limits the reaction; hence, this