Application of Saturation Labeling in Lung Cancer Proteomics - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 255

2.2. Cysteine-Specifi c
Protein Labeling Using
CyDye DIGE Fluor
Saturation Dyes

1. Dye stock solution: CyDye DIGE fl uor saturation dyes (GE
Healthcare, Life Sciences, Freiburg, Germany). The dyes are
reconstituted in high-quality anhydrous dimethylformamide
(DMF), giving a concentration of 2 mM (100 nmol dye in 50 L

DMF) for analytical gels and 20 mM (400 nmol dye in
20

μ

L DMF) for preparative gels. The dye solutions are stable
at −20°C for 3 months.
2. 2 mM Triscarboxethylphosphine (TCEP) solution for analytical
gels and 20 mM TCEP solution for preparative gels: prepare
freshly.
3. 1.4 M DTT: store in aliquots at −20°C.
4. Image software: ImageQuant™ TL v2005 (GE Healthcare).
5. Differential image analysis software: Decyder 2D 6.0 (GE
Healthcare).
6. Fluorescence Scanner: Typhoon 9400 (GE Healthcare).

μ

1. Servalyt 2-4 (Serva, Heidelberg, Germany): store at 4°C.
2. Separation gel buffer: 3.59% (w/v) acrylamide, 0.31% (w/v)
piperazindiacrylamide, 4.1% (v/v) carrier ampholines mixture
(pH 2-11), 9.0 M urea, 5.1% (w/w) glycerol, and 0.06% (v/v)
TEMED: store in aliquots at −80°C.
3. Cap gel buffer: 12.3% (w/v) acrylamide, 0.13% (w/v) piper-
azindiacrylamide, 4.1% (v/v) carrier ampholyte mixture, 9.0 M
urea, 5.1% (w/w) glycerol, and 0.06% (v/v) TEMED: store in
aliquots at −80°C.
4. 1.2% (w/w) Ammonium persulfate (APS): store in aliquots at
−80°C.
5. Anodic buffer: 3 M urea and 0.74 M phosphoric acid: prepare
freshly.
6. Cathodic buffer: 9 M urea, 5% (w/v) glycerol, and 0.75 M
ethylenediamine: prepare freshly.
7. Sephadex solution: 270 mg Sephadex suspension (20 g
Sephadex swollen in 500 mL of water and resuspended into
1 L of 25% (v/v) glycerol solution and fi ltered) with 233 mg
urea, 98 mg thiourea, 25

2.3. Isoelectric
Focusing

μ L of ampholine mixture, pH 2-11,

L of 1.4 M DTT: store in aliquots at −20°C.
8. Protection solution: 30% (w/v) urea, 5% (w/w) glycerol, and
2% carrier ampholytes, pH 2-4: store in aliquots at −80°C.
9. Equilibration solution: 125 mM Tris base, 40% (w/w) glycerol,
65 mM DTT, and 104 mM SDS: store in aliquots at −20°C.

and 25 μ

2.4. Two-Dimensional
Polyacrylamide Gel
Electrophoresis

1. Glass plates compatible with fl uorescence imaging

(25 × 30 × 0.4 cm).
2. Plastic spacer (30 × 1 × 0.15 cm).