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 1st and 2nd dimension
disruption
protocols
7 M urea,
3 mg/ml sample lysate in 1st Dimension
2 M thiourea,
water. Sample precipitated 24 cm, pH 3-10 NL
30 mM Tris,
using acetone on ice for 1 Immobiline DryStrip.
4% CHAPS
h.
(pH 8.5).
Centrifuged at 4 °C
Ettan IPGphor IEF unit,
(12 000 g, 10 min).
anodic cup loading.
Supernatant discarded and
pellet resuspended in lysis
50 µA per strip.
buffer. Lysate
1. 300 V, 3 h, step.
concentration 2.5 mg/ml
2. 600 V, 3 h, gradient.
prior to labelling.
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.
.
25µg 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 1st and 2nd dimension
disruption
protocols
7 M urea,
Whole flies mechanically 1st Dimension
2 M thiourea,
homogenized, directly in 24 cm, pH 3-10 NL
25 mM Tris,
lysis buffer. Incubated
Immobiline DryStrip
4% CHAPS
on ice for 1 h. Centrifuged
(pH 8.0–8.5).
at 4 °C (12 000 g, 20
Ettan IPGphor IEF unit,
min). Pellet discarded and anodic cup loading.
supernatant used for
labelling.
50 µA 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 µg 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 1st and 2nd dimension
disruption
protocols
7 M urea,
Lysis buffer added to cell 1st Dimension
2 M thiourea,
pellet. Sonicated on wet 24 cm, pH 3-10 NL
30 mM Tris,
ice with low-intensity 30 s Immobiline DryStrip.
4% CHAPS
pulses until the lysate
(pH 8.5).
turned clear. Centrifuged Ettan IPGphor IEF unit,
at 4 °C (12 000 g, 10
cathodic cup loading.
min). Pellet discarded and
supernatant used for
50 µA per strip.
labelling.
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 µg 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 1st and 2nd dimension
disruption
protocols
7 M urea,
Lysis buffer added to cell 1st Dimension
2 M thiourea,
pellet. Sonicated on wet 24 cm, pH 4-5 Immobiline
30 mM Tris,
ice with low-intensity 30 s DryStrip.
4% CHAPS
pulses until the lysate
(pH 8.5).
turned clear. Centrifuged Ettan IPGphor IEF unit,
at 4 °C (12 000 g, 10
anodic cup loading.
min). Pellet discarded and
supernatant used for
50 µA per strip.
labelling.
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 µg 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 1st and 2nd dimension
disruption
protocols
7 M urea,
Lysis buffer added to cell 1st Dimension
2 M thiourea,
pellet. Sonicated on wet 24 cm, pH 4.5-5.5
30 mM Tris,
ice with low-intensity 30 s Immobiline DryStrip.
4% CHAPS
pulses until the lysate
(pH 8.5).
turned clear. Centrifuged Ettan IPGphor IEF unit,
at 4 °C (12 000 g, 10
anodic cup loading.
min). Pellet discarded and
supernatant used for
50 µA per strip.
labelling.
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 µg 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 1st and 2nd dimension
disruption
protocols
7 M urea,
Lysis buffer added to cell 1st Dimension
2 M thiourea,
pellet. Sonicated on wet 24 cm, pH 4-7 Immobiline
30 mM Tris,
ice with low-intensity 30 s DryStrip.
4% CHAPS
pulses until the lysate
(pH 8.5).
turned clear. Centrifuged Ettan IPGphor IEF unit,
at 4 °C (12 000 g, 10
anodic cup loading.
min). Pellet discarded and
supernatant used for
50 µA per strip.
labelling.
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 µg 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 1st and 2nd dimension
disruption
protocols
7 M urea,
Lysis buffer added to cell 1st Dimension
2 M thiourea,
pellet. Sonicated on wet 24 cm, pH 5-6 Immobiline
30 mM Tris,
ice with low-intensity 30 s DryStrip.
4% CHAPS
pulses until the lysate
(pH 8.5).
turned clear. Centrifuged Ettan IPGphor IEF unit,
at 4 °C (12 000 g, 10
anodic cup loading.
min). Pellet discarded and
supernatant used for
50 µA per strip.
labelling.
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 µg 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 Method of cell or tissue
disruption
disruption
7 M urea,
Lysis buffer added to cell 1st Dimension
2 M thiourea,
pellet. Sonicated on wet 24 cm, pH 5.5-6.7
30 mM Tris,
ice with low-intensity 30 s Immobiline DryStrip.
4% CHAPS
pulses until the lysate
(pH 8.5).
turned clear. Centrifuged Ettan IPGphor IEF unit,
at 4 °C (12 000 g, 10
anodic cup loading.
min). Pellet discarded and
supernatant used for
50 µA per strip.
labelling.
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 µg 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 1st and 2nd dimension
disruption
protocols
7 M urea,
Lysis buffer added to cell 1st Dimension
2 M thiourea,
pellet. Sonicated on wet 24 cm, pH 6-9 Immobiline
30 mM Tris,
ice with low-intensity 30 s DryStrip.
4% CHAPS
pulses until the lysate
DeStreak reagent used.
(pH 8.5).
turned clear. Centrifuged
at 4 °C (12 000 g, 10
Ettan IPGphor IEF unit,
min). Pellet discarded and anodic cup loading.
supernatant used for
labelling.
50 µA 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 µg 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
8 M urea,
Sample diluted directly in
40 mM Tris,
lysis buffer to 10 mg/ml.
4% CHAPS
(pH 8.0).
1st and 2nd dimension
protocols
1st Dimension
24 cm, pH 3-10 NL
Immobiline DryStrip.
Ettan IPGphor IEF unit,
anodic cup loading.
50 µA 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 µg 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 1st and 2nd dimension
disruption
protocols
8 M urea,
Washed 4× with saline
1st Dimension
30 mM Tris,
solution (0.9%, 10 ml).
24 cm, pH 3-10 NL
4% CHAPS
Saline solution drained.
Immobiline DryStrip.
(pH 8.5).
Cut into small pieces, lysis
buffer added and
Ettan IPGphor IEF unit,
mechanically homogenized anodic cup loading.
at room temperature.
Centrifuged at 4 °C (13
50 µA per strip.
000 rpm, 10 min). Pellet
1. 300 V, 3 h, step.
discarded and supernatant
2. 600 V, 3 h, gradient.
used for labelling.
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 µg 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 1st and 2nd dimension
disruption
protocols
7 M urea,
Washed 4× with saline
1st Dimension
2 M thiourea,
solution (0.9%, 10 ml).
24 cm, pH 3-10 NL
30 mM Tris,
Saline solution drained.
Immobiline DryStrip.
4% CHAPS
Cut into small pieces, lysis
(pH 8.5).
buffer added and
Ettan IPGphor IEF unit,
mechanically homogenized anodic cup loading.
at room temperature.
Centrifuged at 4 °C (13
50 µA per strip.
000 rpm, 10 min). Pellet
1. 300 V, 3 h, step.
discarded and supernatant
2. 600 V, 3 h, gradient.
used for labelling.
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 µg 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 1st and 2nd dimension
disruption
protocols
7 M urea,
Mechanically homogenized 1st Dimension
2 M thiourea,
in ice-cold lysis buffer.
24 cm, pH 3-10 NL
30 mM Tris,
Centrifuged at 4 °C (13
Immobiline DryStrip.
4% CHAPS
000 rpm, 10 min). Pellet
(pH 8.5).
discarded and supernatant Ettan IPGphor IEF unit,
used for labelling.
anodic cup loading.
50 µA 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 µg 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 1st and 2nd dimension
disruption
protocols
8 M urea,
Washed 4× with saline
1st Dimension
30 mM Tris,
solution (0.9%, 10 ml).
24 cm, pH 3-10 NL
4% CHAPS
Saline solution drained.
Immobiline DryStrip.
(pH 8.5).
Cut into small pieces, lysis
buffer added and
Ettan IPGphor IEF unit,
mechanically homogenized anodic cup loading.
at room temperature.
Centrifuged at 4 °C (13
50 µA per strip.
000 rpm, 10 min). Pellet
1. 300 V, 3 h, step.
discarded and supernatant
2. 600 V, 3 h, gradient.
used for labelling.
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 µg 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 1st and 2nd dimension
disruption
protocols
7 M urea,
Washed 4× with saline
1st Dimension
2 M thiourea,
solution (0.9%, 10 ml).
24 cm, pH 3-10 NL
30 mM Tris,
Saline solution drained.
Immobiline DryStrip.
4% CHAPS
Cut into small pieces.
(pH 8.5).
Lysis buffer added and
Ettan IPGphor IEF unit,
mechanically homogenized anodic cup loading.
at room temperature.
Centrifuged at 4 °C (13
50 µA per strip.
000 rpm, 10 min). Pellet
1. 300 V, 3 h, step.
discarded and supernatant
2. 600 V, 3 h, gradient.
used for labelling.
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 µg 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 1st and 2nd dimension
disruption
protocols
7 M urea,
Trypsinized cells washed 1st Dimension
2 M thiourea,
twice with wash buffer and 24 cm, pH 3-10 NL
30 mM Tris,
diluted 1 in 10 with lysis Immobiline DryStrip.
4% CHAPS,
buffer. Sample sonicated
5 mM magnesium on wet ice with
Ettan IPGphor IEF unit,
acetate
low-intensity 30 s pulses anodic cup loading.
(pH 8.5).
until the lysate turned
clear. Centrifuged at 4 °C
50 µA per strip.
(12 000 g, 10 min). Pellet
1. 300 V, 3 h, step.
discarded and supernatant
2. 600 V, 3 h, gradient.
used for labelling.
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 µg 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 1st and 2nd dimension
disruption
protocols
7 M urea,
1 g of tissue placed in
1st Dimension
2 M thiourea,
10 ml of lysis buffer.
24 cm, pH 4-7 Immobiline
10 mM Tris,
Tissue
DryStrip.
5 mM magnesium mechanically homogenized
acetate,
and then centrifuged at
Ettan IPGphor IEF unit,
4% CHAPS
10 °C (12 000 g, 1 h).
anodic cup loading.
(pH 8.0).
Pellet discarded and
supernatant used for
50 µA per strip.
labelling.
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 µg 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 1st and 2nd dimension
disruption
protocols
7 M urea,
1 g of tissue placed in
1st Dimension
2 M thiourea,
10 ml of lysis buffer.
24 cm, pH 3-10 NL
10 mM Tris,
Tissue
Immobiline DryStrip.
5 mM magnesium mechanically homogenized
acetate,
and then centrifuged at
Ettan IPGphor IEF unit,
4% CHAPS
10 °C (12 000 g, 1 h).
anodic cup loading.
(pH 8.0).
Pellet discarded and
supernatant used for
50 µA per strip.
labelling.
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 µg 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 1st and 2nd dimension
disruption
protocols
7 M urea,
Washed 3× with saline
1st Dimension
2 M thiourea,
solution and drained. Cut 24 cm, pH 3-10 NL
30 mM Tris,
into small pieces, lysis
Immobiline DryStrip.
5 mM magnesium buffer added and
acetate,
mechanically homogenized Ettan IPGphor IEF unit,
4% CHAPS
at room temperature.
anodic cup loading.
(pH 8.5).
Centrifuged at 4 °C (13
000 rpm, 10 min). Pellet
50 µA per strip.
discarded and supernatant
1. 300 V, 3 h, step.
used for labelling.
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 µg 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 1st and 2nd dimension
disruption
protocols
7 M urea,
8ml of plasma mixed with 1st Dimension
2 M thiourea,
10ml of lysis buffer.
24 cm, pH 3-10 NL
30 mM Tris,
Centrifuged at 10 °C
Immobiline DryStrip.
5 mM magnesium (12 000 g, 1 h). Pellet
acetate,
discarded and supernatant Ettan IPGphor IEF unit,
4% CHAPS
used for labelling. Lysate anodic cup loading.
(pH 8.0).
concentration 10.9 mg/ml
prior to labelling.
50 µA 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 µg 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 1st and 2nd dimension
disruption
protocols
7 M urea,
Dried cell preparation
1st Dimension
2 M thiourea,
resuspended in lysis
24 cm, pH 3-10 NL
30 mM Tris,
buffer.
Immobiline DryStrip.
4% CHAPS
Sonicated on wet ice with
(pH 8.5).
low-intensity 30 s pulses Ettan IPGphor IEF unit,
until the lysate turned
anodic cup loading.
clear. Centrifuged at 4 °C
(12 000 g, 5 min). Pellet
50 µA per strip.
discarded and supernatant
1. 300 V, 3 h, step.
used for labelling.
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 µg 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 1st and 2nd dimension
disruption
protocols
7 M urea,
Medium was removed,
1st Dimension
2 M thiourea,
cells washed twice in PBS 24 cm, pH 3-10 NL
10 mM Tris,
and scraped from the
Immobiline DryStrip.
5 mM magnesium flasks. Cells centrifuged
acetate,
and pellets washed twice Ettan IPGphor IEF unit,
4% CHAPS
in wash buffer (10 mM
anodic cup loading.
(pH 8.0).
Tris, pH 8, 5 mM
magnesium acetate).
50 µA per strip.
Pellets resuspended in
1. 300 V, 3 h, step.
lysis buffer.
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 µg of protein labelled with CyDye DIGE Fluor, Cy5 minimal
dye.