Name: Fusarium graminearum strain 453 reference map

Description: Proteome reference map of the nivalenol producer strain Fusarium graminearum 453 using a 2D-DiGE protocol. In order to obtain a more complete map, the Fusarium graminearum 453 strain was grown in two different culture media, one containing Glutamic acid as source of Nitrogen and the other containing Agmatin as Nitrogen source. Both media are considered to be good inducer of toxin production.

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2010-09-18

1.1.2 Responsible person or role

Affiliation: Centre de Recherce Public Gabriel Lippmann

(i) Name: Dr. Tommaso Serchi

(ii) Postal address: 41, rue du Brill, L4422 Belvaux, Luxembourg

(iii) Email address: serchi@lippmann.lu

1.1.3 Electrophoresis type

Difference gel electrophoresis

2. Sample

2.1.1 Sample Name(s)

    1. Sample name: Fusarium graminearum (strain453) mycelium, isolated in Luxembourg
    2. Sample type: Standard

2.1.2 Loading buffer

  1. 2 M thiourea, 7 M urea, 4% w/v CHAPS, 30 mM Tris pH 8.5, DeStreak reagent (GE Healthecare) 0.005% v/v, 0.2% v/v IPG pH 3-10 (BioRad) buffer, and a trace of bromophenol blue

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: BioRad
Model: ReadyStrip IPG Strips, pH 3–10 nonlinear, 24 cm,
Model number: 163-2043
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

Non Linear pH 3 - 10

3.2.5 Acrylamide concentration

4 %

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 32.5:1

3.2.7 Additional substances in gel

No additional substance

3.2.8 Gel lane

1

3.2.9 Sample application

Lane 1

  • Sample: Fusarium graminearum (strain453) mycelium, isolated in Luxembourg
  • Volume of sample: 90 µg
  • Loading buffer: 2 M thiourea, 7 M urea, 4% w/v CHAPS, 30 mM Tris pH 8.5, DeStreak reagent (GE Healthecare) 0.005% v/v, 0.2% v/v IPG pH 3-10 (BioRad) buffer, and a trace of bromophenol blue
  • Volume of loading buffer: 500 µL

Loading method: rehydration loading.

3.3 Protocol

3.3.1 Buffers

No buffer.

3.3.2 Electrophoresis conditions

Running temperature: 25 °C

Hold: 30 V, 3 h

Gradient: 30-200 V, 2 h

Hold: 200 V, 3 h

Gradient: 200-1000 V, 4 h

Hold: 1000 V, 2 h

Gradient: 1000-5000 V, 4 h

Hold: 5000 V, 1 h

Gradient: 5000-10000 V, 4 h

Hold: 10000 V, 6 h

4. Inter-dimension Process

4.1 Protocol

4.1.1 Step name

reduction

4.1.2 Inter dimension buffer

The buffer used in the reduction is provided by Serva Electrophoresis. The buffer needs to be added 7.2 g of Urea and 200 mg of DTT per 20 mL of equilibration solutuion. The solution provided is already supplemented with bromophenol blue.

4.1.3 Additional reagents

Urea (approximately 6 M)
DTT (approximately 54 mM)

4.1.4 Equipment

4.1.5 Protocol

Temperature: 25 °C.

Duration: 15 min.

Protocol:
Put the strip in a tray face up and add 10 mL of equilibration solution (10 mL per strip). Shacke gently.

4.1.1 Step name

alkylation

4.1.2 Inter dimension buffer

The buffer used in the reduction is provided by Serva Electrophoresis. The buffer needs to be added 7.2 g of Urea and 500 mg of IAA per 20 mL of euilibration solutuion. The solution provided is already supplemented with bromophenol blue.

4.1.3 Additional reagents

Urea (approximately 6 M)
Iodio-Acetamide (approximately 140 mM)

4.1.4 Equipment

4.1.5 Protocol

Temperature: 25 °C.

Duration: 15 min.

Protocol:
Put the strip in a tray face up and add 10 mL of equilibration solution (10 mL per strip). Shacke gently.

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: Serva Electrophoresis
Model: 2D Gel DALTtwelve 12.5 % Kit
Model number: 43319.01
Batch number: unkown

3.2.3 Physical dimensions

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

3.2.4 Physiochemical property range and distribution

linear apparent molecular mass 5 - 200 kDa

3.2.5 Acrylamide concentration

12.5 %

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 32.5:1

3.2.7 Additional substances in gel

No additional substance

3.2.8 Gel lane

1

3.2.9 Sample application

Loading method: Loading method: IPG transfer..

3.3 Protocol

3.3.1 Buffers

The buffers are provided by the Serva Electrophoresis company. They consist of two bags containing one the dry powder for the cathode and one the dry powder for the anode. Dilution was performed following the manufacturer's instruction: briefly, the cathode buffer is to be diluted in 2.5 L of milliQ water; the anode buffer is to be diluted in 7.5 L of milliQ water.

3.3.2 Electrophoresis conditions

Running temperature: 25 °C

Hold: 10 mA, 2 h

Hold: 30 mA, 14 h

5. Detection

5.1 Direct detection

5.1.1 Name of direct detection_process

Fluorescent staining

5.1.2 Direct detection agents

Cy2, Cy3 and Cy5 - CyDyes minimal labeling kit (25 nmol)- GE Healthcare

5.1.3 Additional reagents and buffers

DMF 100%
10 mM Lysin

5.1.4 Equipment

Manufacturer: GE Healthcare
Model: Typhoon 9400
Model number: unknown

5.1.5 Direct detection protocol

Temperature: 20 °C.

Duration: 1 h.

Protocol:
1) A volume of sample equivalent to 30 μg of proteins was added to a microfuge tube.
2) 240 pmol of the appropriate dye were added to the microfuge tube containing the protein sample.
3) Dye and protein sample were mixed thoroughly by pipetting and vortexing.
4) The tube was then centrifuged briefly in a microcentrifuge to collect the solution at the bottom of the tube.
5) The labeling reaction was carried out leaving the tube on ice for 30 min in the dark.
6) The reaction was stopped adding 1 μl of 10 mM lysine to the reaction tube.

6. Image Acquisition

6.1 Acquisition Equipment

6.1.1 Type of equipment

laser scanner

6.1.2 Name of equipment

Manufacturer: GE Healthcare
Model: Typhoon 9400
Model number: unknown

6.1.3 Software

Manufacturer: GE Healthcare
Model: Typhoon Scanner Control
Model number: unknown

6.1.4 Calibration

Yes (automatic)

6.1.5 Equipment specific parameters

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

6.2 Acquisition Protocol

6.2.1 Image acquisition process

1) Turn on the Typhoon and leave the instrument to
warm up for at least 30 min prior to scanning.
2) Ensure that the gel glass plates are clean, dry and free from lint.
3)Select scan area
4)Select the appropriate wavelenths and adjust the intensity of the lasers in a way to have the maximum signal with no saturation. The adjustemnt can be done using a scanning resolution of 1mm
5)After calibration scan the gel setting the resolution to 100 um.

6.2.2 Reference to gel matrix

There is only one gel in this document.

7. Image

7.1.1 Image name (or id)

Cy3 (format: TIFF)

7.1.2 Dimensions

Width: 741 px

Height: 1000 px

7.1.3 Resolution

254 ppi

7.1.4 Bit-depth

8-bit (256 colors)

7.1.5 Image location

Cy3.tiff

7.1.6 Standard image orientation

Yes

7. Image

7.1.1 Image name (or id)

Cy5 (format: TIFF)

7.1.2 Dimensions

Width: 741 px

Height: 1000 px

7.1.3 Resolution

254 ppi

7.1.4 Bit-depth

8-bit (256 colors)

7.1.5 Image location

Cy5.tiff

7.1.6 Standard image orientation

Yes

7. Image

7.1.1 Image name (or id)

Cy2 (format: TIFF)

7.1.2 Dimensions

Width: 741 px

Height: 1000 px

7.1.3 Resolution

254 ppi

7.1.4 Bit-depth

8-bit (256 colors)

7.1.5 Image location

CY2.tiff

7.1.6 Standard image orientation

Yes

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