サンプル調製例（Saturation dyes）

Extraction

Tissue washed with saline (0.9 %), mechanically homogenised in cell lysis buffer (7 M urea, 2 M thiourea, 4 % CHAPS, 40 mM Tris, pH 8.0, 1 ml per 0.1 g tissue) and centrifuged (13,000 rpm, 10 min, 4 ℃). Pellet discarded and supernatant used for labelling.

Labelling

5 μg protein labelled with 4 nmol TCEP and 8 nmol dye.

1st Dimension

pH 3-10 NL, 24 cm Immobiline™ DryStrips.

Ettan™ IPGphor™ IEF unit, anodic cup loading.

50 μA per strip
1. 300 V, 3 h, step
2. 600 V, 3 h, gradient
3. 1,000 V, 3 h, gradient
4. 8,000 V, 3 h, gradient
5. 8,000 V, 4 h, step

2nd Dimension

12.5 % Ettan™ DALT gel,
2 W per gel overnight, 15 ℃.

Extraction

Serum-free medium was poured off and cells washed twice with PBS in the flask. Without trypsinisation, cell lysis buffer (2 M thiourea, 7 M urea, 4 % CHAPS, 40 mM Tris, pH 8.0) was added to the flask. Cell lysate was pipetted out and sonicated on wet ice, with low-intensity 30 s pulses until the lysate turned clear. The sample was centrifuged (13,000 rpm, 10 min, 4 ℃), the pellet discarded and the supernatant used for labelling.

Labelling

5 μg protein labelled with 2 nmol TCEP and 4 nmol dye.

1st Dimension

pH 3-10 NL, 24 cm Immobiline™ DryStrips.

Ettan™ IPGphor™ IEF unit, anodic cup loading.

50 μA per strip
1. 300 V, 3 h, step
2. 600 V, 3 h, gradient
3. 1,000 V, 3 h, gradient
4. 8,000 V, 3 h, gradient
5. 8,000 V, 4 h, step

2nd Dimension

12.5 % Ettan™ DALT gel,
2 W per gel overnight, 15 ℃.

Extraction

Tissue washed 4× with saline (0.9 %) and mechanically homogenised in cell lysis buffer (8 M urea, 4 % CHAPS, 30 mM Tris, pH 8.0, 1 ml per 0.1 g tissue). The supernatant was extracted and sonicated on wet ice, with low-intensity 30 s pulses until the lysate turned clear. The sample was centrifuged (13,000 rpm, 10 min, 4 ℃), the pellet discarded and the supernatant used for labelling.

Labelling

5 μg protein labelled with 2 nmol TCEP and 4 nmol dye

1st Dimension

pH 3-10 NL, 24 cm Immobiline™ DryStrips.

Ettan™ IPGphor™ IEF unit, anodic cup loading.

50 μA per strip
1. 300 V, 3 h, step
2. 600 V, 3 h, gradient
3. 1,000 V, 3 h, gradient
4. 8,000 V, 3 h, gradient
5. 8,000 V, 4 h, step

2nd Dimension

12.5 % Ettan™ DALT gel,
2 W per gel overnight, 15 ℃.

Extraction

Tissue washed 4× with saline (0.9 %) and mechanically homogenised in cell lysis buffer (8 M urea, 4 % CHAPS, 30 mM Tris, pH 8.0, 1 ml per 0.1 g tissue). The supernatant was extracted and sonicated on wet ice, with low-intensity 30 s pulses until the lysate turned clear. The sample was centrifuged (13,000 rpm, 10 min, 4 ℃), the pellet discarded and the supernatant used for labelling.

Labelling

5 μg protein labelled with 2 nmol TCEP and 4 nmol dye.

1st Dimension

pH 3-10 NL, 24 cm Immobiline™ DryStrips.

Ettan™ IPGphor™ IEF unit, anodic cup loading.

50 μA per strip
1. 300 V, 3 h, step
2. 600 V, 3 h, gradient
3. 1,000 V, 3 h, gradient
4. 8,000 V, 3 h, gradient
5. 8,000 V, 4 h, step

2nd Dimension

12.5 % Ettan™ DALT gel,
2 W per gel overnight, 15 ℃.