Synthesis and Validation of Cyanine-Based Dyes for DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 68

all samples. The same amount of the Cy2-labeled standard is then
loaded on all the gels in the experiment along with individual
samples labeled with Cy3 and Cy5. The ratios of Cy3-to-Cy2 and
Cy5-to-Cy2 can then be calculated and normalized against the
Cy2-labeled internal standard ( 3 ). This approach resulted in fur-
ther elimination of technical noise in DIGE experiments and
enabled the analysis of the relative abundances for each protein
spot resolved on gels with high precision and unprecedented statis-
tical power ( 4, 5 ).
The DIGE method was originally introduced in 1997 ( 2 ); in a
few years, it was commercialized by GE Healthcare, which offered
a full line of reagents and equipment necessary to successfully
implement the technology, including the dyes for labeling. Besides
being commercially available, the dyes could be synthesized using
known procedures ( 2, 6, 7 ). In this chapter, we provide detailed
procedures for the effi cient synthesis of all three dyes used in DIGE,
benzoxazolium dye Cy2, and two indocyanine dyes, propyl Cy3
and ethyl Cy5, and their NHS esters, from commercially available
precursors. We also validated the newly synthesized dyes in DIGE
experiments by comparing them to commercially available dyes
and evaluated the effects of long-term and short-term storage of
the synthesized dyes on DIGE output.

2. Materials

2.1. Dye Synthesis

The following reagents were obtained from Sigma-Aldrich:
2-methylbenzoxazole, bromoethane, 1,2-dichlorobenzene, 4-(bromo-
methyl) phenylacetic acid, N,N ¢-diphenylformamidine, N,N ¢-dis-
uccini-midyl carbonate (DSC), 2,3,3-trimethyl-3 H -indole,
1-bromopropane, 6-bromohexanoic acid, malondialdehyde
bis(dimethyl acetal), 1,3,3-trimethyl-2-methyleneindoline.
Other reagents and common solvents were obtained from
Fisher Scientifi c: anhydrous N,N -dimethylformamide (DMF), diethyl
ether, acetonitrile, acetic anhydride, dichloromethane, hexane,
methanol, absolute ethanol, anhydrous triethylamine, anhydrous
pyridine, glacial acetic acid, anhydrous sodium acetate, and HCl.
Aniline was obtained from Alfa Aesar.

The following reagents and materials were supplied by GE Health-
care: urea, thiourea, CHAPS, 2D Quant kit, CyDye fl uor minimal
dyes Cy2, Cy3, and Cy5, and IPG strips. Other reagents were obtained
from Sigma-Aldrich (DMF, SDS, DTT, iodoacetamide, L -lysine,
magnesium acetate), and Fisher Scientifi c (glycerol, isopropanol).
All 2D electrophoresis equipment, the IPGPhor II (fi rst-
dimension isoelectric focusing) and the DALT 12 apparatus (sec-
ond-dimension SDS-PAGE) as well as the DeCyder v. 6.5 software
for DIGE analysis were obtained from GE Healthcare.

2.2. DIGE Experiments