2D DIGE Analysis of Serum After Fractionation by ProteoMiner Beads - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 184

4. DTT: 2 M DTT in water. Store aliquots at −20°C.

5. Immobiline DryStrip cover fl uid (GE Healthcare).

2.6. Second-
Dimension SDS PAGE

1. Equilibration buffer: 6 M urea, 30% (v/v) glycerol, 50 mM

Tris-HCl (pH 8.8), 2% (v/v) SDS, containing trace amount of
bromophenol blue. Store at −20°C in aliquots of 10 mL.

2. 40% Acrylamide/bis solution (37.5:1 with 2.6% C) and

TEMED (Bio-Rad).

3. 0.75% (w/v) agarose overlay: to make 100 mL of solution, use

microwave oven to melt 0.75 g of low-melting agarose in 100 mL
of 1× SDS-running buffer. Add trace amount of bromophenol
blue and store in 2 mL aliquots at room temperature.

4. DTT.

5. Iodoacetamide (IAA).

2.7. Silver Staining

1. Fixing solution: 50% (v/v) methanol, 12% (v/v) acetic acid,

and 0.05% (v/v) formalin.

2. Wash solution: 35% (v/v) ethanol in water.

3. Sensitizing solution: 0.02% (w/v) sodium thiosulfate in water.

4. Silver nitrate solution: 0.2% (w/v) silver nitrate and 0.076%

(v/v) formalin.

5. Developing solution: 6% (w/v) sodium carbonate, 0.004%

(w/v) sodium thiosulfate, and 0.05% (v/v) formalin.

6. Stop solution: 1.46% (w/v) sodium-EDTA in water.

2.8. Image Analysis

DeCyder™ 2D Differential Analysis Software.

3. Methods

All biological samples should be handled in a Class II biohazard
hood. All waste generated should be decontaminated with 10%
(v/v) bleach or autoclaved before discarding.

3.1. DIGE Experimental
Design

Firstly, the experimental design is planned to determine the amount
of sample required to be cleaned up for optimization and the actual
DIGE experiment. Table 1 outlines the experimental design for
labeling proteins for four sera samples. A total of 150 mg protein
is loaded on each gel with a pooled sample as the internal standard.
The internal standard in all the gels is composed of an equal
aliquot of every sample in the entire DIGE experiment. The internal
standard facilitates protein spots matching on different gels and
allows relative protein abundance to be calculated. Dye swap is