Comparative 2D DIGE Analysis of the Depleted Serum Proteome for Biomarker Discovery - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 209

2.1. Depletion
of High-Abundance
Proteins

1. MARS-M3 column (4.6 × 100 mm; Agilent Technologies,
Santa Clara, CA, USA). Store at 4°C and maintain at room
temperature when in use.
2. Spin fi lters, 0.22-mm pore size cellulose acetate (Agilent
Technologies).
3. MARS buffer A and buffer B (Agilent Technologies).
4. HP 1090 HPLC system (Hewlett Packard, Palo Alto, CA, USA).
5. 15- and 50-mL Falcon polypropylene tubes (BD Biosciences,
San Jose, CA, USA).
6. 100% acetone, stored at −20°C in a solvent-compatible freezer.

2.2. Sample
Preparation for DIGE

1. 80% acetone, stored at −20°C in a solvent-compatible freezer.
2. TUC4 buffer: 7 M urea, 2 M thiourea, 30 mM Tris-HCl , 4%

(w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-
sulfonate (CHAPS), and pH 8.5. Stored at −20°C in 1 mL
aliquots.
3. 10 kDa MWCO Vivaspin 500 centrifugal devices (GE
Healthcare).
4. Twin Cond B-173 conductivity meter (Horiba, Kyoto, Japan).
5. Gel-loading pipette tips.
6. EZQ protein quantitation assay (Invitrogen, Carlsbad,
CA, USA).

1. Cy2, Cy3, and Cy5 DIGE fl uor CyDyes (GE Healthcare):
working solutions of 200 pmol CyDye per 1 mL of anhydrous
99.8% N , N -dimethylformamide. Store 1 mL aliquots at −80°C
(see Note 1). Thaw on ice when required, protected from
light.
2. 10 mM L -lysine. Store in aliquots at −20°C.
3. 1 M dithiothreitol (DTT). Store aliquots at −80°C and thaw at
room temperature when required.
4. IPG buffer pH 4-7 (GE Healthcare). Store at 4°C.

2.3. DIGE Labeling

2.4. Two-Dimensional
Electrophoresis (2DE)

1. Rehydration buffer: 7 M urea, 2 M thiourea, 30 mM Tris-HCl,
2% (w/v) CHAPS, 0.5% (v/v) IPG buffer, 1.2% (v/v) DeStreak
reagent (GE Healthcare; store at 4°C), and 0.05% (v/v) satu-
rated bromophenol blue solution (prepared in water). IPG buf-
fer, DeStreak, and bromophenol blue can be added freshly to
1 mL aliquots of TUC2 buffer (7 M urea, 2 M thiourea, 30 mM
Tris-HCl, 2% (w/v) CHAPS), which can be stored at −20°C.
2. 24-cm IPG strips, pH 4-7 (GE Healthcare). Store at −20°C.
3. PlusOne dry strip cover fl uid (GE Healthcare).
4. Ettan IPGphor II (GE Healthcare).