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Name: fnsinip47g63403

Description: Insoluble fraction : comparison of C. elegans proteins from worms infected or not by the fungus Drechmeria coniospora

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2008-07-15

1.1.2 Responsible person or role

Affiliation: Centre d'Immunologie de Marseille Luminy

(i) Name: Dr Jonathan Ewbank

(ii) Postal address: Research group of Innate immunity in C. elegans

Case 906
163 avenue de luminy
13288 Marseille Cedex 9
France

(iii) Email address: ewbank@ciml.univ-mrs.fr

1.1.3 Electrophoresis type

Two-Dimensional electrophoresis

2. Sample

2.1.1 Sample Name(s)

    1. Sample name: 25 µg of infected C. elegans proteins Cy3 labeled , 25 %g of naive C. elegans proteins Cy5 labeled , 25 µg internal standard Cy2 labeled
    2. Sample type: Test sample
    1. Sample name: 25 µg of infected C. elegans proteins Cy3 labeled , 25 %g of naive C. elegans proteins Cy5 labeled , 25 µg internal standard Cy2 labeled
    2. Sample type: Test sample
    1. Sample name: 25 µg of naive C. elegans proteins Cy3 labeled , 25 µg of infected C. elegans proteins Cy5 labeled , 25 µg internal standard Cy2 labeled
    2. Sample type: Test sample
    1. Sample name: 25 µg of naive C. elegans proteins Cy3 labeled , 25 µg of infected C. elegans proteins Cy5 labeled , 25 µg internal standard Cy2 labeled
    2. Sample type: Test sample

2.1.2 Loading buffer

  1. Urea 7M Thiourea 2M Chaps 4% ampholyte 1%

3. Gel matrix and electrophoresis protocol

3.1 Dimension details

3.1.1 Ordinal number for this dimension

First

3.1.2 Separation method employed

Isoelectric focusing (IEF)

3.2 Gel Matrix

3.2.1 Description of gel matrix

IPG strip
Denaturing

3.2.2 Gel manufacture

Gel was purchased precast.

Manufacturer: GE-healtcare
Model: ImmobilineTM DryStrip pH 4-7 linear 11 cm strips
Model number: 18-1016-60
Batch number: 63403-63404-63405-63407

3.2.3 Physical dimensions

X: 108 mm
Y: 3 mm
Z: 0.5 mm

3.2.4 Physiochemical property range and distribution

linear pH 4 - 7

3.2.5 Acrylamide concentration

4 %

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 3:1

3.2.7 Additional substances in gel

Destreak rehydration solution: ref 17-6003-19 GE Healthcare

3.2.8 Gel lane

1

3.2.9 Sample application

Lane 1

  • Sample: 25 µg of infected C. elegans proteins Cy3 labeled , 25 %g of naive C. elegans proteins Cy5 labeled , 25 µg internal standard Cy2 labeled
  • Volume of sample: 75 µg
  • Loading buffer: Urea 7M Thiourea 2M Chaps 4% ampholyte 1%
  • Volume of loading buffer: 35 µL

Loading method: cup loading.

3.3 Protocol

3.3.1 Buffers

No buffer.

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Gradient: 0-30 V, 3 h

Gradient: 30-300 V, 3 h

Gradient: 300-8000 V, 8 h

Hold: 8000 V, 6 h

4. Inter-dimension Process

4.1 Protocol

4.1.1 Step name

equilibration

4.1.2 Inter dimension buffer

6 M Urea; 50 mM Tris-HCl pH 8,8; 34.5 %(v/v)Glycerol; 2 %(v/v)SDS; 65mM DTT; trace of Bromophenol blue

4.1.3 Additional reagents

No additional reagent.

4.1.4 Equipment

4.1.5 Protocol

Temperature: 20 °C.

Duration: 20 min.

Protocol:
20 minutes in equilibration buffer

3. Gel matrix and electrophoresis protocol

3.1 Dimension details

3.1.1 Ordinal number for this dimension

Second

3.1.2 Separation method employed

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)

3.2 Gel Matrix

3.2.1 Description of gel matrix

slab gel
Denaturing

3.2.2 Gel manufacture

Gel was purchased precast.

Manufacturer: Bio-rad
Model: criterion XT bis-tris gel 10%
Model number: 3450115
Batch number: unknown

3.2.3 Physical dimensions

X: 133 mm
Y: 87 mm
Z: 1 mm

3.2.4 Physiochemical property range and distribution

logarithmic apparent molecular mass 10 - 250 kDa

3.2.5 Acrylamide concentration

10 %

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 10:5

3.2.7 Additional substances in gel

No additional substance

3.2.8 Gel lane

1

3.2.9 Sample application

Loading method: well loading.

3.3 Protocol

3.3.1 Buffers

MOPS 1x

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Hold: 10 mA, 30 min

Hold: 30 mA, 30 min

Hold: 60 mA, 105 min

5. Detection

5.1 Direct detection

5.1.1 Name of direct detection_process

Fluorescent staining

5.1.2 Direct detection agents

GE Healtcare cy dye DIGE Fluor minimal labelling kit ref: 25-8010-65
Cy3, Cy5, Cy2 : 200 pmol cydye per sample

5.1.3 Additional reagents and buffers

DMF 100%
Lysine 10mmol

5.1.4 Equipment

No specialised equipment.

5.1.5 Direct detection protocol

Temperature: 4 °C.

Duration: 1 h.

Protocol:
Each Cy Dye is solved in 5 µl of 100% DMF as a stock solution. 1µl of the stock solution is diluted in 4µl of 100% DMF as the labelling solution. 1 µl of the Cy3 or the Cy5 Dye solution is added separatly to 25 µg protein of each "test" and each "control" sample. 4 µl of the Cy2 solution is added to the internal standard protein mixture, which contains 12.5 µg protein from each sample. Minimal labeling is done for 30 min at 4°C in the dark. Then, 1 µl of the lysine solution is added to each sample to stop the labeling reaction during 10 min still at 4°C in the dark. Each sample are combined according to the design experimental (see section 2.1.1), and DTT is added at a final concentration of 10 mmol for further 10 minutes at 4°C in the dark. The combined samples were supplemented with an equal volume of 2x sample buffer (8M urea, 2M thiourea, 4% (w/v) CHAPS, 1% (v/v) IPG Buffer 4-7 (GE Healthcare)

6. Image Acquisition

6.1 Acquisition Equipment

6.1.1 Type of equipment

fluorescent scanner

6.1.2 Name of equipment

Manufacturer: GE-healtcare
Model: Ettan Dige Imager
Model number: 11-0036-58

6.1.3 Software

Manufacturer: GE-healtcare
Model: EDI software
Model number: 1.0

6.1.4 Calibration

Yes (automatic)

6.1.5 Equipment specific parameters

Excitation and emission filters combinations are automatically selected by the software:
Cy2 excitation filter 480/30 nm and emission filter 530/40 nm
Cy3 excitation filter 540/25 nm and emission filter 595/25 nm
Cy5 excitation filter 635/30 nm and emission filter 680/25 nm

6.2 Acquisition Protocol

6.2.1 Image acquisition process

exposure time settings
gel1
0.4
0.15
0.2
gel2
1
0.5
0.7
gel3
0.6
0.2
0.3
gel4
0.4
0.15
0.2






















6.2.2 Reference to gel matrix

There is only one gel in this document.

7. Image

7.1.1 Image name (or id)

fnsinip47g63403 (format: JPEG)

7.1.2 Dimensions

Width: 1097 px

Height: 655 px

7.1.3 Resolution

100 µm/px

7.1.4 Bit-depth

24-bit (TrueColor)

7.1.5 Image location

fnsinip47g63403.jpg

7.1.6 Standard image orientation

Yes

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