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Name: C. elegans - S. aureus 4 hours

Description: The C. elegans proteome 4 hours after infection with S. aureus

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2009-09-16

1.1.2 Responsible person or role

Affiliation: Katholieke Universiteit Leuven

(i) Name: Prof. Dr. Liliane Schoofs

(ii) Postal address: Department of Biology
Research group of Functional Genomics and Proteomics
Naamsestraat 59
3000 Leuven
Belgium

(iii) Email address: Liliane.Schoofs@bio.kuleuven.be

1.1.3 Electrophoresis type

Difference gel electrophoresis

2. Sample

2.1.1 Sample Name(s)

    1. Sample name: 75µg of naive C. elegans proteins labeled with Cy3, 75µg of immune-challenged C. elegans proteins labeled with Cy5, 75 µg of internal standard labeled with Cy2
    2. Sample type: Test sample
    1. Sample name: 75µg of naive C. elegans proteins labeled with Cy5, 75µg of immune-challenged C. elegans proteins labeled with Cy3, 75 µg of internal standard labeled with Cy2
    2. Sample type: Test sample
    1. Sample name: 75µg of naive C. elegans proteins labeled with Cy3, 75µg of immune-challenged C. elegans proteins labeled with Cy5, 75 µg of internal standard labeled with Cy2
    2. Sample type: Test sample
    1. Sample name: 75µg of naive C. elegans proteins labeled with Cy5, 75µg of immune-challenged C. elegans proteins labeled with Cy3, 75 µg of internal standard labeled with Cy2
    2. Sample type: Test sample
    1. Sample name: 75µg of naive C. elegans proteins labeled with Cy3, 75µg of immune-challenged C. elegans proteins labeled with Cy5, 75 µg of internal standard labeled with Cy2
    2. Sample type: Test sample
    1. Sample name: 75µg of naive C. elegans proteins labeled with Cy5, 75µg of immune-challenged C. elegans proteins labeled with Cy3, 75 µg of internal standard labeled with Cy2
    2. Sample type: Test sample

2.1.2 Loading buffer

  1. Urea (7 M) Thiourea (2 M) 4% CHAPS 40 mM Tris 1% DDT

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 Healthcare Bio-Sciences Corp.
Model: ImmobilineTM DryStrip pH 3-10 Non-linear 24 cm strips
Model number: 17-6002-45
Batch number: unknown

3.2.3 Physical dimensions

X: 240 mm
Y: 3 mm
Z: 0.5 mm

3.2.4 Physiochemical property range and distribution

sigmoidal pH 3 - 10

3.2.5 Acrylamide concentration

4 %

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 32:1

3.2.7 Additional substances in gel

Destreak rehydration solution (GE Healthcare, 17-6003-19)

3.2.8 Gel lane

1

3.2.9 Sample application

Lane 1

  • Sample: 75µg of naive C. elegans proteins labeled with Cy3, 75µg of immune-challenged C. elegans proteins labeled with Cy5, 75 µg of internal standard labeled with Cy2
  • Volume of sample: 225 µg
  • Loading buffer: Urea (7 M) Thiourea (2 M) 4% CHAPS 40 mM Tris 1% DDT
  • Volume of loading buffer: 50 µL

Loading method: cup loading.

3.3 Protocol

3.3.1 Buffers

No buffer.

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Hold: 150 V, 3 h

Hold: 300 V, 3 h

Hold: 1000 V, 6 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

Buffer A: 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 50 mM Tris-HCl (pH 8.8) and 1% (w/v) DTT in the first step and 4% (w/v) iodoacetamide
Buffer B: buffer A in which DTT was replaced with 4% (w/v) iodoacetamide

4.1.3 Additional reagents

A trace of bromophenol blue in the second equilibration step

4.1.4 Equipment

Manufacturer: GE Healthcare Bio-Sciences Corp.
Model: Immobiline Drystrip Reswelling Tray
Model number: 80-6465-32

4.1.5 Protocol

Temperature: 20 °C.

Duration: 30 min.

Protocol:
15 min in buffer A followed by 15 min in buffer B

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 manufactured using the following receipe:

30 % Acrylamide/Bis (250 ml), Tris-Cl pH 8.8 (150 ml), Double distilled water (187 ml), 10% SDS (6 ml), 10% APS (6 ml), 10% TEMED (830 µl)

3.2.3 Physical dimensions

X: 260 mm
Y: 200 mm
Z: 1 mm

3.2.4 Physiochemical property range and distribution

logarithmic apparent molecular mass 10 - 200 kDa

3.2.5 Acrylamide concentration

12.48 %

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 37.5:1

3.2.7 Additional substances in gel

No additional substances

3.2.8 Gel lane

1

3.2.9 Sample application

Loading method: IPG transfer.

3.3 Protocol

3.3.1 Buffers

3 X SDS
1 X SDS

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Hold: 8 mA, 1 h

Hold: 12 mA, 12 h

5. Detection

5.1 Direct detection

5.1.1 Name of direct detection_process

Fluorescent staining

5.1.2 Direct detection agents

Cy dyes: Cy3, Cy5, Cy2 (GE Healtcare: 5 nmol CyDye DIGE Fluor (minimal Dye) Labelling Kit), 0.267 nmol per Cy Dye per sample (see 2.1.1)

5.1.3 Additional reagents and buffers

100% DMF, 0,128% (w/v) lysine

5.1.4 Equipment

Manufacturer: unknown
Model: no specialised equipment
Model number: unknown

5.1.5 Direct detection protocol

Temperature: 20 °C.

Duration: 1 h.

Protocol:
Cy Dye is solved in 12,5 µl of 100% DMF, 2 µl of the Cy3 or the Cy5 Dye solution is added separatly to 75 µg protein of each test and each control sample. 12 µl of the Cy2 Due solution is added to the internal standard protein mixture, which contains 75 µg protein from each sample. Minimal labeling is done for 45 min at 4°C in the dark. Then, 1 µl of the lysine solution added to each sample to stop the labeling reaction (10 min 4°C in the dark).

6. Image Acquisition

6.1 Acquisition Equipment

6.1.1 Type of equipment

fluorescent scanner

6.1.2 Name of equipment

Manufacturer: GE Healthcare Bio-Sciences Corp.
Model: Ettan DIGE Imager
Model number: unknown

6.1.3 Software

Manufacturer: GE Healthcare Bio-Sciences Corp.
Model: Ettan TM DIGE Imager Software
Model number: unknown

6.1.4 Calibration

Yes (automatic)

6.1.5 Equipment specific parameters

Cy3 excitation filter 540/25 nm and emission filter 595/25 nm
Cy5 excitation filter 635/30 nm and emission filter 680/25 nm
Cy2 excitation filter 480/30 nm and emission filter 530/40 nm

6.2 Acquisition Protocol

6.2.1 Image acquisition process

UNKNOWN

6.2.2 Reference to gel matrix

There is only one gel in this document.

7. Image

7.1.1 Image name (or id)

Ce_Sa_4h (format: BMP)

7.1.2 Dimensions

Width: 490 px

Height: 465 px

7.1.3 Resolution

96 dpi

7.1.4 Bit-depth

32-bit (TrueColor)

7.1.5 Image location

C:\Documents and Settings\Annelies\Desktop\Ce_Sa_4h.bmp

7.1.6 Standard image orientation

Yes

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