Name: C. elegans proteome after infection with Staphylococcus aureus

Description: C. elegans proteome after infection with the gram-negative bacterium Aeromonas hydrophila

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

Two-Dimensional electrophoresis

2. Sample

2.1.1 Sample Name(s)

    1. Sample name: proteome of C. elegans challenged with S. aureus
    2. Sample type: Control 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: proteome of C. elegans challenged with S. aureus
  • Volume of sample: 300 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

Silver staining

5.1.2 Direct detection agents

0.1 % silver nitrate

5.1.3 Additional reagents and buffers

1. 50% methanol and 5% acetic acid
2. 50% methanol
3. milli-Q
4. 0.02% sodium thiosulfate
5. 2% sodium carbonate and 0,04% formaldehyde
6. 5% acetic acid
7. 1% acetic acid

5.1.4 Equipment

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

5.1.5 Direct detection protocol

Temperature: 20 °C.

Duration: 1.5 h.

Detection is described in the following reference protocol:
Citation: Anal. Chem.,68, page(s) 850-858 (1996).
URL: not provided.

6. Image Acquisition

6.1 Acquisition Equipment

6.1.1 Type of equipment

camera

6.1.2 Name of equipment

Manufacturer: Nikon
Model: Nikon Coolpix 990
Model number: unknown

6.1.3 Software

Manufacturer: unknown
Model: no software used
Model number: unknown

6.1.4 Calibration

Yes (automatic)

6.1.5 Equipment specific parameters

unknown

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_silver (format: TIFF)

7.1.2 Dimensions

Width: 1000 px

Height: 1000 px

7.1.3 Resolution

500 dpi

7.1.4 Bit-depth

8-bit (256 colors)

7.1.5 Image location

C:\Documents and Settings\Annelies\Desktop\CE-SA paper\grijs_zonder_tekst-1000x1000.tif

7.1.6 Standard image orientation

Yes

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