Proteomic Profiling of the Epithelial-Mesenchymal Transition Using 2D DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 275

2.2. CyDye
Reconstitution and
Protein Labeling

Throughout the protocol, MilliQ-quality water should be used for
making all aqueous solutions, and the pH adjusted using either
5 M HCl or 1 M NaOH, where appropriate:

1. CyDye DIGE fl uor, Cy2 minimal dye (GE Healthcare, Cat no:
RPK0272).
2. CyDye DIGE fl uor, Cy3 minimal dye (GE Healthcare, Cat no:
RPK0273).
3. CyDye DIGE fl uor, Cy5 minimal dye (GE Healthcare, Cat no:
RPK0275).
4. Dimethylformamide (DMF).
5. Lysine.

1. IPG Buffer pH 3-10 (GE Healthcare, Cat no: 17-6000-87).
2. DeStreak rehydration solution (GE Healthcare, Cat no:
17-6003-19).
3. Immobiline DryStrip re-swelling tray (GE Healthcare, Cat no:
80-6465-32).
4. Immobiline DryStrip pH 3-10, 24 cm (GE Healthcare, Cat
no: 17-6002-44).
5. Immobiline DryStrip cover fl uid (GE Healthcare, Cat no:
17-1335-01).
6. Ettan IPGphor 3 isoelectric focusing unit (GE Healthcare, Cat
no: 11-0033-64).
7. Paper electrode pads (GE Healthcare, Cat no: 80-6499-14).
8. IPGphor manifold ceramic tray (GE Healthcare, Cat no:
80-6498-57).
9. Electrode set (GE Healthcare, Cat no: 80-6498-76).
10. Sample cups (GE Healthcare, Cat no: 80-6498-95).
11. Equilibration buffer: 6 M urea, 30% (v/v) glycerol, 2% (w/v)
SDS, 50 mM Tris-HCl, pH 8.8.
12. Dithiothreitol (DTT).
13. Iodoacetamide.

2.3. Immobiline DryStrip
Rehydration and IEF

2.4. Polyacrylamide
Gel Casting and
SDS-PAGE

1. Low-fl uorescence glass plates for Ettan DALT (GE Healthcare,
Cat no: 80-6475-58).
2. DALT twelve Gel Caster Complete (GE Healthcare, Cat no:
80-6467-22).
3. DALT gradient maker with peristaltic pump (GE Healthcare,
Cat no: 80-6067-84).
4. 8% light acrylamide solution: 120 mL of 30% (w/v) acrylam-
ide, 113 mL of 1.5 M Tris-HCl, pH 8.8, 207 mL of water,
4.5 mL of 10% (w/v) SDS, 4.5 mL of 10% ammonium persulfate
(APS), 0.96 mL of 10% TEMED.