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サンプル調製例(Saturation dyes)

Saturation dyes

Saturation dyesを使用した2Dパターンおよびサンプル調製例(タンパク質抽出法、標識の方法、二次元電気泳動のプロトコール)を以下に示します。

Sample Types

Mouse brain

Extraction and Labelling Protocols

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 and 2nd Dimension Conditions

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 ℃.

Mouse brainのゲルイメージ
Cy3/Cy5 overlay for 5 μg protein labelled with CyDye DIGE Fluor Cy3 saturation dye (red) and 5 μg protein labelled with CyDye DIGE Fluor Cy5 saturation dye (blue).

HEP G2 cultured cell line

Extraction and Labelling Protocols

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 and 2nd Dimension Conditions

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 ℃.

HEP G2 cultured cell lineのゲルイメージ
Cy3/Cy5 overlay for 5 μg protein labelled with CyDye DIGE Fluor Cy3 saturation dye (red) and 5 μg protein labelled with CyDye DIGE Fluor Cy5 saturation dye (blue).

Rat liver

Extraction and Labelling Protocols

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 and 2nd Dimension Conditions

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 ℃.

Rat liverのゲルイメージ
Cy3/Cy5 overlay for 5 μg protein labelled with CyDye DIGE Fluor Cy3 saturation dye (red) and 5 μg protein labelled with CyDye DIGE Fluor Cy5 saturation dye (blue).

Rat lung

Extraction and Labelling Protocols

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 and 2nd Dimension Conditions

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 ℃.

Rat lungのゲルイメージ
Cy3/Cy5 overlay for 5 μg protein labelled with CyDye DIGE Fluor Cy3 saturation dye (red) and 5 μg protein labelled with CyDye DIGE Fluor Cy5 saturation dye (blue).


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