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Sample Specific Protocols for Minimal Dyes and Saturation Dyes

Sample Specific Protocols for Minimal Dyes

Sample Specific Protocols for
Saturation Dyes

Sample Specific Protocols for Minimal Dyes
The following set of tables show protocols which have been used for a wide range of sample types, alongside examples of the 2-D images obtained.

All protein lysates were labelled at a concentration of 5–10 mg/ml unless otherwise stated.

The protocols used here are not necessarily optimal methods for these sample types but do present a useful methodology along with an illustration of the image quality that can be obtained in each case.

All IEF programs used finished with a low voltage (500 V) step for 48 h. This step was intended to maintain the focusing of the proteins after the IEF program had completed. The number of hours spent at this voltage varied for each sample type but was generally significantly lower than the full 48 h programmed into the IEF unit.

Strips were removed immediately upon completion of the IEF program. Where this was not possible and samples were left at 500 V for more than 2 h, they were then refocused by ramping to 8000 V over a period of 30 min.

Sample Types
Canorhabditis elegans (C.elegans), pH 3-10 NL IPG strip
Drosophila melanogaster (D. melanogaster), pH 3-10 NL IPG strip
Escherichia coli (E.coli) cell culture, pH 3-10 NL IPG strip
Escherichia coli (E.coli) cell culture, pH 4-5 IPG strip
Escherichia coli (E.coli) cell culture, pH 4.5-5.5 IPG strip
Escherichia coli (E.coli) cell culture, pH 4-7 IPG strip
Escherichia coli (E.coli) cell culture, pH 5-6 IPG strip
Escherichia coli (E.coli) cell culture, pH 5.5-6.7 IPG strip
Escherichia coli (E.coli) cell culture, pH 6-9 IPG strip
Human serum, pH 3-10 NL IPG strip
Mouse cerebellum, pH 3-10 NL IPG strip, 8 M urea lysis buffer
Mouse cerebellum, pH 3-10 NL IPG strip, 7 M urea, 2 M thiourea lysis buffer
Mouse striatum, pH 3-10 NL IPG strip
Mouse skeletal muscle, pH 3-10 NL IPG strip, 8 M urea lysis buffer
Mouse skeletal muscle, pH 3-10 NL IPG strip, 7 M urea, 2 M thiourea lysis buffer
NIH 3T3 fibroblasts, pH 3-10 NL IPG strip
Rat heart, pH 3-10 NL IPG strip
Rat liver, pH 3-10 NL IPG strip
Rat Kidney, pH 3-10 NL IPG strip
Rat plasma, pH 3-10 NL IPG strip
Saccharomyces cerevisiae (S. cerevisiae), pH 3-10 NL IPG strip
Bladder epithelial carcinoma T24 cell line, pH 3-10 NL IPG strip

Canorhabditis elegans (C.elegans), pH 3-10 NL IPG strip.

Lysis buffer	Method of cell or tissue disruption	1st and 2nd dimension protocols
7 M urea, 2 M thiourea, 30 mM Tris, 4% CHAPS (pH 8.5).	3 mg/ml sample lysate in water. Sample precipitated using acetone on ice for 1 h. Centrifuged at 4 �C (12 000 g, 10 min). Supernatant discarded and pellet resuspended in lysis buffer. Lysate concentration 2.5 mg/ml prior to labelling.	1st Dimension 24 cm, pH 3-10 NL Immobiline DryStrip. Ettan IPGphor IEF unit, anodic cup loading. 50 �A per strip. 1. 300 V, 3 h, step. 2. 600 V, 3 h, gradient. 3. 1000 V, 3 h, gradient. 4. 8000 V, 3 h, gradient. 5. 8000 V, 4 h, step. 2nd Dimension 12.5% Ettan DALTtwelve 1.5 W per gel overnight. .
25�g of protein labelled with CyDye DIGE Fluor, Cy5 minimal dye.
.. *Drosophila melanogaster* (**D. melanogaster), pH 3-10 NL IPG strip.**
Lysis buffer	Method of cell or tissue disruption	1st and 2nd dimension protocols
7 M urea, 2 M thiourea, 25 mM Tris, 4% CHAPS (pH 8.0–8.5).	Whole flies mechanically homogenized, directly in lysis buffer. Incubated on ice for 1 h. Centrifuged at 4 �C (12 000 g, 20 min). Pellet discarded and supernatant used for labelling.	1st Dimension 24 cm, pH 3-10 NL Immobiline DryStrip Ettan IPGphor IEF unit, anodic cup loading. 50 �A per strip. 1. 300 V, 3 h, step. 2. 600 V, 3 h, gradient. 3. 1000 V, 3 h, gradient. 4. 8000 V, 3 h, gradient. 5. 8000 V, 4 h, step. 2nd Dimension 12.5% Ettan DALTtwelve 1.5 W per gel overnight. .
50 �g protein labelled with CyDye DIGE Fluor, Cy5 minimal dye.
..
*Escherichia* *coli* (**E.coli) cell culture, pH 3-10 NL IPG strip.**
Lysis buffer	Method of cell or tissue disruption	1st and 2nd dimension protocols
7 M urea, 2 M thiourea, 30 mM Tris, 4% CHAPS (pH 8.5).	Lysis buffer added to cell pellet. Sonicated on wet ice with low-intensity 30 s pulses until the lysate turned clear. Centrifuged at 4 �C (12 000 g, 10 min). Pellet discarded and supernatant used for labelling.	1st Dimension 24 cm, pH 3-10 NL Immobiline DryStrip. Ettan IPGphor IEF unit, cathodic cup loading. 50 �A per strip. 1. 500 V, 1 h, step. 2. 1000 V, 1 h, step. 3. 8000 V, 8 h, step. 2nd Dimension 12.5% Ettan DALTtwelve 2 W per gel overnight. .
50 �g of protein labelled with CyDye DIGE Fluor, Cy5 minimal dye.
..
*Escherichia coli* (**E.coli) cell culture, pH 4-5 IPG strip.**
Lysis buffer	Method of cell or tissue disruption	1st and 2nd dimension protocols
7 M urea, 2 M thiourea, 30 mM Tris, 4% CHAPS (pH 8.5).	Lysis buffer added to cell pellet. Sonicated on wet ice with low-intensity 30 s pulses until the lysate turned clear. Centrifuged at 4 �C (12 000 g, 10 min). Pellet discarded and supernatant used for labelling.	1st Dimension 24 cm, pH 4-5 Immobiline DryStrip. Ettan IPGphor IEF unit, anodic cup loading. 50 �A per strip. 1. 300 V, 3 h, step. 2. 600 V, 3 h, gradient. 3. 1000 V, 3 h, gradient. 4. 8000 V, 3 h, gradient. 5. 8000 V, 4 h, step. 2nd Dimension 12.5% Ettan DALTtwelve 2 W per gel overnight. .
50 �g of protein labelled with CyDye DIGE Fluor, Cy3 minimal dye.
..
*Escherichia coli* (**E.coli) cell culture, pH 4.5-5.5 IPG strip.**
Lysis buffer	Method of cell or tissue disruption	1st and 2nd dimension protocols
7 M urea, 2 M thiourea, 30 mM Tris, 4% CHAPS (pH 8.5).	Lysis buffer added to cell pellet. Sonicated on wet ice with low-intensity 30 s pulses until the lysate turned clear. Centrifuged at 4 �C (12 000 g, 10 min). Pellet discarded and supernatant used for labelling.	1st Dimension 24 cm, pH 4.5-5.5 Immobiline DryStrip. Ettan IPGphor IEF unit, anodic cup loading. 50 �A per strip. 1. 300 V, 3 h, step. 2. 600 V, 3 h, gradient. 3. 1000 V, 3 h, gradient. 4. 8000 V, 3 h, gradient. 5. 8000 V, 4 h, step. 2nd Dimension 12.5% Ettan DALTtwelve 2 W per gel overnight. .
50 �g of protein labelled with CyDye DIGE Fluor, Cy3 minimal dye.
..
*Escherichia coli* (**E.coli) cell culture, pH 4-7 IPG strip.**
Lysis buffer	Method of cell or tissue disruption	1st and 2nd dimension protocols
7 M urea, 2 M thiourea, 30 mM Tris, 4% CHAPS (pH 8.5).	Lysis buffer added to cell pellet. Sonicated on wet ice with low-intensity 30 s pulses until the lysate turned clear. Centrifuged at 4 �C (12 000 g, 10 min). Pellet discarded and supernatant used for labelling.	1st Dimension 24 cm, pH 4-7 Immobiline DryStrip. Ettan IPGphor IEF unit, anodic cup loading. 50 �A per strip. 1. 300 V, 3 h, step. 2. 600 V, 3 h, gradient. 3. 1000 V, 3 h, gradient. 4. 8000 V, 3 h, gradient. 5. 8000 V, 4 h, step. 2nd Dimension 12.5% Ettan DALTtwelve 2 W per gel overnight. .
50 �g of protein labelled with CyDye DIGE Fluor, Cy3 minimal dye.
..
*Escherichia coli* (**E.coli) cell culture, pH 5-6 IPG strip.**
Lysis buffer	Method of cell or tissue disruption	1st and 2nd dimension protocols
7 M urea, 2 M thiourea, 30 mM Tris, 4% CHAPS (pH 8.5).	Lysis buffer added to cell pellet. Sonicated on wet ice with low-intensity 30 s pulses until the lysate turned clear. Centrifuged at 4 �C (12 000 g, 10 min). Pellet discarded and supernatant used for labelling.	1st Dimension 24 cm, pH 5-6 Immobiline DryStrip. Ettan IPGphor IEF unit, anodic cup loading. 50 �A per strip. 1. 300 V, 3 h, step. 2. 600 V, 3 h, gradient. 3. 1000 V, 3 h, gradient. 4. 8000 V, 3 h, gradient. 5. 8000 V, 4 h, step. 2nd Dimension 12.5% Ettan DALTtwelve 2 W per gel overnight. .
50 �g of protein labelled with CyDye DIGE Fluor, Cy3 minimal dye.
..
*Escherichia coli* (**E.coli) cell culture, pH 5.5-6.7 IPG strip.**
Lysis buffer	Method of cell or tissue disruption	Method of cell or tissue disruption
7 M urea, 2 M thiourea, 30 mM Tris, 4% CHAPS (pH 8.5).	Lysis buffer added to cell pellet. Sonicated on wet ice with low-intensity 30 s pulses until the lysate turned clear. Centrifuged at 4 �C (12 000 g, 10 min). Pellet discarded and supernatant used for labelling.	1st Dimension 24 cm, pH 5.5-6.7 Immobiline DryStrip. Ettan IPGphor IEF unit, anodic cup loading. 50 �A per strip. 1. 300 V, 3 h, step. 2. 600 V, 3 h, gradient. 3. 1000 V, 3 h, gradient. 4. 8000 V, 3 h, gradient. 5. 8000 V, 4 h, step. 2nd Dimension 12.5% Ettan DALTtwelve 2 W per gel overnight. .
50 �g of protein labelled with CyDye DIGE Fluor, Cy3 minimal dye.
..
*Escherichia coli* (**E.coli) cell culture, pH 6-9 IPG strip.**
Lysis buffer	Method of cell or tissue disruption	1st and 2nd dimension protocols
7 M urea, 2 M thiourea, 30 mM Tris, 4% CHAPS (pH 8.5).	Lysis buffer added to cell pellet. Sonicated on wet ice with low-intensity 30 s pulses until the lysate turned clear. Centrifuged at 4 �C (12 000 g, 10 min). Pellet discarded and supernatant used for labelling.	1st Dimension 24 cm, pH 6-9 Immobiline DryStrip. DeStreak reagent used. Ettan IPGphor IEF unit, anodic cup loading. 50 �A per strip. 1. 300 V, 3 h, step. 2. 600 V, 3 h, gradient. 3. 1000 V, 3 h, gradient. 4. 8000 V, 3 h, gradient. 5. 8000 V, 4 h, step. 2nd Dimension 12.5% Ettan DALTtwelve 2 W per gel overnight. .
50 �g of protein labelled with CyDye DIGE Fluor, Cy3 minimal dye.
..
Human serum, pH 3-10 NL IPG strip.
Lysis buffer	Method of cell or tissue disruption	1st and 2nd dimension protocols
8 M urea, 40 mM Tris, 4% CHAPS (pH 8.0).	Sample diluted directly in lysis buffer to 10 mg/ml.	1st Dimension 24 cm, pH 3-10 NL Immobiline DryStrip. Ettan IPGphor IEF unit, anodic cup loading. 50 �A per strip. 1. 300 V, 3 h, step. 2. 600 V, 3 h, gradient. 3. 1000 V, 3 h, gradient. 4. 8000 V, 3 h, gradient. 5. 8000 V, 4 h, step. 2nd Dimension 12.5% Ettan DALTtwelve 1.5 W per gel overnight. .
50 �g of protein labelled with CyDye DIGE Fluor, Cy3 minimal dye.
..
Mouse cerebellum, pH 3-10 NL IPG strip, 8 M urea lysis buffer.
Lysis buffer	Method of cell or tissue disruption	1st and 2nd dimension protocols
8 M urea, 30 mM Tris, 4% CHAPS (pH 8.5).	Washed 4� with saline solution (0.9%, 10 ml). Saline solution drained. Cut into small pieces, lysis buffer added and mechanically homogenized at room temperature. Centrifuged at 4 �C (13 000 rpm, 10 min). Pellet discarded and supernatant used for labelling.	1st Dimension 24 cm, pH 3-10 NL Immobiline DryStrip. Ettan IPGphor IEF unit, anodic cup loading. 50 �A per strip. 1. 300 V, 3 h, step. 2. 600 V, 3 h, gradient. 3. 1000 V, 3 h, gradient. 4. 8000 V, 3 h, gradient. 5. 8000 V, 4 h, step. 2nd Dimension 12.5% Ettan DALTtwelve 2 W per gel overnight. .
50 �g of protein labelled with CyDye DIGE Fluor, Cy5 minimal dye.
..
Mouse cerebellum, pH 3-10 NL IPG strip, 7 M urea, 2 M thiourea lysis buffer.
Lysis buffer	Method of cell or tissue disruption	1st and 2nd dimension protocols
7 M urea, 2 M thiourea, 30 mM Tris, 4% CHAPS (pH 8.5).	Washed 4� with saline solution (0.9%, 10 ml). Saline solution drained. Cut into small pieces, lysis buffer added and mechanically homogenized at room temperature. Centrifuged at 4 �C (13 000 rpm, 10 min). Pellet discarded and supernatant used for labelling.	1st Dimension 24 cm, pH 3-10 NL Immobiline DryStrip. Ettan IPGphor IEF unit, anodic cup loading. 50 �A per strip. 1. 300 V, 3 h, step. 2. 600 V, 3 h, gradient. 3. 1000 V, 3 h, gradient. 4. 8000 V, 3 h, gradient. 5. 8000 V, 4 h, step. 2nd Dimension 12.5% Ettan DALTtwelve 2 W per gel overnight. .
50 �g of protein labelled with CyDye DIGE Fluor, Cy5 minimal dye.
..
Mouse striatum, pH 3-10 NL IPG strip.
Lysis buffer	Method of cell or tissue disruption	1st and 2nd dimension protocols
7 M urea, 2 M thiourea, 30 mM Tris, 4% CHAPS (pH 8.5).	Mechanically homogenized in ice-cold lysis buffer. Centrifuged at 4 �C (13 000 rpm, 10 min). Pellet discarded and supernatant used for labelling.	1st Dimension 24 cm, pH 3-10 NL Immobiline DryStrip. Ettan IPGphor IEF unit, anodic cup loading. 50 �A per strip. 1. 300 V, 3 h, step. 2. 600 V, 3 h, gradient. 3. 1000 V, 3 h, gradient. 4. 8000 V, 3 h, gradient. 5. 8000 V, 4 h, step. 2nd Dimension 12.5% Ettan DALTtwelve 2 W per gel overnight. .
50 �g of protein labelled with CyDye DIGE Fluor, Cy5 minimal dye

Mouse skeletal muscle, pH 3-10 NL IPG strip, 8 M urea lysis buffer.
Lysis buffer	Method of cell or tissue disruption	1st and 2nd dimension protocols
8 M urea, 30 mM Tris, 4% CHAPS (pH 8.5).	Washed 4� with saline solution (0.9%, 10 ml). Saline solution drained. Cut into small pieces, lysis buffer added and mechanically homogenized at room temperature. Centrifuged at 4 �C (13 000 rpm, 10 min). Pellet discarded and supernatant used for labelling.	1st Dimension 24 cm, pH 3-10 NL Immobiline DryStrip. Ettan IPGphor IEF unit, anodic cup loading. 50 �A per strip. 1. 300 V, 3 h, step. 2. 600 V, 3 h, gradient. 3. 1000 V, 3 h, gradient. 4. 8000 V, 3 h, gradient. 5. 8000 V, 4 h, step. 2nd Dimension 12.5% Ettan DALTtwelve 1.5 W per gel overnight. .
50 �g of protein labelled with CyDye DIGE Fluor, Cy5 minimal dye.
..
Mouse skeletal muscle, pH 3-10 NL IPG strip, 7 M urea, 2 M thiourea lysis buffer.
Lysis buffer	Method of cell or tissue disruption	1st and 2nd dimension protocols
7 M urea, 2 M thiourea, 30 mM Tris, 4% CHAPS (pH 8.5).	Washed 4� with saline solution (0.9%, 10 ml). Saline solution drained. Cut into small pieces. Lysis buffer added and mechanically homogenized at room temperature. Centrifuged at 4 �C (13 000 rpm, 10 min). Pellet discarded and supernatant used for labelling.	1st Dimension 24 cm, pH 3-10 NL Immobiline DryStrip. Ettan IPGphor IEF unit, anodic cup loading. 50 �A per strip. 1. 300 V, 3 h, step. 2. 600 V, 3 h, gradient. 3. 1000 V, 3 h, gradient. 4. 8000 V, 3 h, gradient. 5. 8000 V, 4 h, step. 2nd Dimension 12.5% Ettan DALTtwelve 2 W per gel overnight. .
50 �g of protein labelled with CyDye DIGE Fluor, Cy5 minimal dye.
..
NIH 3T3 fibroblasts, pH 3-10 NL IPG strip.
Lysis buffer	Method of cell or tissue disruption	1st and 2nd dimension protocols
7 M urea, 2 M thiourea, 30 mM Tris, 4% CHAPS, 5 mM magnesium acetate (pH 8.5).	Trypsinized cells washed twice with wash buffer and diluted 1 in 10 with lysis buffer. Sample sonicated on wet ice with low-intensity 30 s pulses until the lysate turned clear. Centrifuged at 4 �C (12 000 g, 10 min). Pellet discarded and supernatant used for labelling.	1st Dimension 24 cm, pH 3-10 NL Immobiline DryStrip. Ettan IPGphor IEF unit, anodic cup loading. 50 �A per strip. 1. 300 V, 3 h, step. 2. 600 V, 3 h, gradient. 3. 1000 V, 3 h, gradient. 4. 8000 V, 3 h, gradient. 5. 8000 V, 4 h, step. 2nd Dimension 12.5% Ettan DALTtwelve 1.5 W per gel overnight. .
50 �g of protein labelled with CyDye DIGE Fluor, Cy5 minimal dye.
..
Rat heart, pH 3-10 NL IPG strip.
Lysis buffer	Method of cell or tissue disruption	1st and 2nd dimension protocols
7 M urea, 2 M thiourea, 10 mM Tris, 5 mM magnesium acetate, 4% CHAPS (pH 8.0).	1 g of tissue placed in 10 ml of lysis buffer. Tissue mechanically homogenized and then centrifuged at 10 �C (12 000 g, 1 h). Pellet discarded and supernatant used for labelling.	1st Dimension 24 cm, pH 4-7 Immobiline DryStrip. Ettan IPGphor IEF unit, anodic cup loading. 50 �A per strip. 1. 300 V, 3 h, step. 2. 600 V, 3 h, gradient. 3. 1000 V, 3 h, gradient. 4. 8000 V, 3 h, gradient. 5. 8000 V, 4 h, step. 2nd Dimension 12.5% Ettan DALTtwelve 1.5 W per gel overnight. .
50 �g of protein labelled with CyDye DIGE Fluor, Cy5 minimal dye.
..
Rat liver, pH 3-10 NL IPG strip.
Lysis buffer	Method of cell or tissue disruption	1st and 2nd dimension protocols
7 M urea, 2 M thiourea, 10 mM Tris, 5 mM magnesium acetate, 4% CHAPS (pH 8.0).	1 g of tissue placed in 10 ml of lysis buffer. Tissue mechanically homogenized and then centrifuged at 10 �C (12 000 g, 1 h). Pellet discarded and supernatant used for labelling.	1st Dimension 24 cm, pH 3-10 NL Immobiline DryStrip. Ettan IPGphor IEF unit, anodic cup loading. 50 �A per strip. 1. 300 V, 3 h, step. 2. 600 V, 3 h, gradient. 3. 1000 V, 3 h, gradient. 4. 8000 V, 3 h, gradient. 5. 8000 V, 4 h, step. 2nd Dimension 12.5% Ettan DALTtwelve 1.5 W per gel overnight. .
50 �g of protein labelled with CyDye DIGE Fluor, Cy5 minimal dye.
..
Rat kidney, pH 3-10 NL IPG strip.
Lysis buffer	Method of cell or tissue disruption	1st and 2nd dimension protocols
7 M urea, 2 M thiourea, 30 mM Tris, 5 mM magnesium acetate, 4% CHAPS (pH 8.5).	Washed 3� with saline solution and drained. Cut into small pieces, lysis buffer added and mechanically homogenized at room temperature. Centrifuged at 4 �C (13 000 rpm, 10 min). Pellet discarded and supernatant used for labelling.	1st Dimension 24 cm, pH 3-10 NL Immobiline DryStrip. Ettan IPGphor IEF unit, anodic cup loading. 50 �A per strip. 1. 300 V, 3 h, step. 2. 600 V, 3 h, gradient. 3. 1000 V, 3 h, gradient. 4. 8000 V, 3 h, gradient. 5. 8000 V, 4 h, step. 2nd Dimension 12.5% Ettan DALTtwelve 1.5 W per gel overnight. .
50 �g of protein labelled with CyDye DIGE Fluor, Cy5 minimal dye.
.
Rat plasma, pH 3-10 NL IPG strip.
Lysis buffer	Method of cell or tissue disruption	1st and 2nd dimension protocols
7 M urea, 2 M thiourea, 30 mM Tris, 5 mM magnesium acetate, 4% CHAPS (pH 8.0).	8ml of plasma mixed with 10ml of lysis buffer. Centrifuged at 10 �C (12 000 g, 1 h). Pellet discarded and supernatant used for labelling. Lysate concentration 10.9 mg/ml prior to labelling.	1st Dimension 24 cm, pH 3-10 NL Immobiline DryStrip. Ettan IPGphor IEF unit, anodic cup loading. 50 �A per strip. 1. 300 V, 3 h, step. 2. 600 V, 3 h, gradient. 3. 1000 V, 3 h, gradient. 4. 8000 V, 3 h, gradient. 5. 8000 V, 4 h, step. 2nd Dimension 12.5% Ettan DALTtwelve 2 W per gel overnight. .
50 �g of protein labelled with CyDye DIGE Fluor, Cy5 minimal dye.
.
*Saccharomyces cerevisiae* (**S. cerevisiae), pH 3-10 NL IPG strip.**
Lysis buffer	Method of cell or tissue disruption	1st and 2nd dimension protocols
7 M urea, 2 M thiourea, 30 mM Tris, 4% CHAPS (pH 8.5).	Dried cell preparation resuspended in lysis buffer. Sonicated on wet ice with low-intensity 30 s pulses until the lysate turned clear. Centrifuged at 4 �C (12 000 g, 5 min). Pellet discarded and supernatant used for labelling.	1st Dimension 24 cm, pH 3-10 NL Immobiline DryStrip. Ettan IPGphor IEF unit, anodic cup loading. 50 �A per strip. 1. 300 V, 3 h, step. 2. 600 V, 3 h, gradient. 3. 1000 V, 3 h, gradient. 4. 8000 V, 3 h, gradient. 5. 8000 V, 4 h, step. 2nd Dimension 12.5% Ettan DALTtwelve 1.5 W per gel overnight. .
50 �g of protein labelled with CyDye DIGE Fluor, Cy5 minimal dye.
.
Bladder epithelial carcinoma T24 cell line, pH 3-10 NL IPG strip.
Lysis buffer	Method of cell or tissue disruption	1st and 2nd dimension protocols
7 M urea, 2 M thiourea, 10 mM Tris, 5 mM magnesium acetate, 4% CHAPS (pH 8.0).	Medium was removed, cells washed twice in PBS and scraped from the flasks. Cells centrifuged and pellets washed twice in wash buffer (10 mM Tris, pH 8, 5 mM magnesium acetate). Pellets resuspended in lysis buffer.	1st Dimension 24 cm, pH 3-10 NL Immobiline DryStrip. Ettan IPGphor IEF unit, anodic cup loading. 50 �A per strip. 1. 300 V, 3 h, step. 2. 600 V, 3 h, gradient. 3. 1000 V, 3 h, gradient. 4. 8000 V, 3 h, gradient. 5. 8000 V, 4 h, step. 2nd Dimension 12.5% Ettan DALTtwelve 1.5 W per gel overnight. .
50 �g of protein labelled with CyDye DIGE Fluor, Cy5 minimal dye.