Synthesis and Validation of Cyanine-Based Dyes for DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 78

copies ( 23 ). Fis has been implicated in transcriptional regulation of
multiple genes ( 24 ); hence, differential expression of many different
proteins may also be expected when WT E. coli and Fis − mutant
cells are analyzed using the DIGE method. This study is designed
to compare individual protein abundances calculated by the DIGE
method after using either LAB or COM dyes for protein labeling.

Five independently grown E. coli WT and fi ve Fis − mutant cultures
were grown in LB for 15 h. A stationary overnight culture was
diluted 1/50 in prewarmed LB medium and incubated for 45 min
at 37°C as Fis protein level is known to reach its maximum at this
time ( 23 ). Immediately after removing the cell cultures from the
incubator, they were quickly chilled on ice and harvested by cen-
trifugation at 10,000 × g for 10 min at 4°C. The pellets were washed
twice with 20 mM Tris-HCl, pH 8.0, 5 mM magnesium acetate.
Cell pellets (from approximately 10 10 cells) were resuspended in
the lysis buffer containing 7 M urea, 2 M thiourea, 4% CHAPS,
20 mM Tris-HCl, pH 8.8, and sonicated on ice for six cycles of
30 s each at high-power setting with 1 min of interruption between
the cycles (see Note 2). The samples were centrifuged at 14,000 × g
for 15 min at 4°C, and the protein concentration was determined
using 2D Quant kit.

3.2.1. Sample Preparation

A total of 50 mg of each protein sample was labeled with 400 pmol
of either Cy3 or Cy5 dyes, standard conditions for a so-called mini-
mal labeling. A pool of 25-mg aliquots collected from each sample
was labeled with 400 pmol Cy2 dye/every 50 mg of protein and
used as an internal standard for each gel. The labeling reaction was
stopped after 30-min incubation on ice by adding 1 mL of 10 mM
L -lysine/400 pmol dye and incubated on ice for additional 10 min.
The rehydration solution containing 7 M urea, 2 M thiourea, 4%
CHAPS, 5% glycerol, 10% isopropanol, 1% DTT, and 0.5% IPG
4-7 buffer was added to the labeled protein samples to a fi nal vol-
ume of 450 mL. The samples were incubated for 20-30 min at
room temperature (RT) and then centrifuged at 12,000 × g for
5 min at RT.

3.2.2. Labeling
with Cy Dyes

Protein samples (450 mL) were loaded overnight onto a 24-cm pH
4-7 IPG strips (GE Healthcare) using an IPGPhor II apparatus
(GE Healthcare). Isoelectric focusing (IEF) was performed at
20°C at 50 mA for a total of 80,000 Vh. After the IEF, IPG strips
were incubated at RT in equilibration buffer containing 50 mM
Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, and 10 mg/
mL DTT for 15 min with gentle shaking, and then in the buffer
containing 50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2%
SDS, and 40 mg/mL iodoacetamide for another 15 min. IPG
strips were then rinsed in the 1× SDS running buffer and placed on
top of the 12.5% SDS-PAGE gels and run at 25 V for 1 h, 50 V for

3.2.3. IEF, SDS-PAGE,
and Scanning