Name: gel448_BY2_ext_chloromethanol_1mg_A2

Description: 2D-gel of 1 mg of soluble proteins from BY2 cells performed on IPG strips (pH 4.0 to 7.0), followed by SDS-PAGE (12% acrylamide)and colloidal Coomassie Blue staining.

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2008-09-16

1.1.2 Responsible person or role

Affiliation: Université Catholique de Louvain-la-Neuve

(i) Name: Duby Geoffrey

(ii) Postal address: Institut des sciences de la vie
Unité FYSA
Place Croix du Sud, 4-5 BTE 15
1348 Louvain-la-Neuve
Belgique

(iii) Email address: geoffrey.duby@uclouvain.be

1.1.3 Electrophoresis type

Two-Dimensional electrophoresis

2. Sample

2.1.1 Sample Name(s)

    1. Sample name: 1 mg of soluble proteins from Nicotiana tabacum Bright- Yellow-2 BY2 cells
    2. Sample type: Test sample

2.1.2 Loading buffer

  1. IEF solubilization buffer : 7 M urea 2 M thiourea 4 % (w/v) CHAPS 0.5 % (v/v) IPG (Immobilized pH Gradient) buffer (pH 4-7) 18 mM DTT 0.002 % (w/v) 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: GE Healthcare
Model: GE Healthcare Immobiline DryStrip pH 4-7, 24 cm
Model number: 17-6002-46
Batch number: unknown

3.2.3 Physical dimensions

X: 235 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: 32.3:1

3.2.7 Additional substances in gel

No additional substance

3.2.8 Gel lane

1

3.2.9 Sample application

Lane 1

  • Sample: 1 mg of soluble proteins from Nicotiana tabacum Bright- Yellow-2 BY2 cells
  • Volume of sample: 1 mg
  • Loading buffer: IEF solubilization buffer : 7 M urea 2 M thiourea 4 % (w/v) CHAPS 0.5 % (v/v) IPG (Immobilized pH Gradient) buffer (pH 4-7) 18 mM DTT 0.002 % (w/v) bromophenol blue
  • Volume of loading buffer: 450 µL

Loading method: rehydration loading.
Additional comment: 2 hours of passive rehydratation at 20°C and 10 hours of active rehydratation at 20°C under 30 V

3.3 Protocol

3.3.1 Buffers

No buffer.

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Gradient: 0-500 V, 2 h

Gradient: 500-1000 V, 2 h

Gradient: 1000-8000 V, 2 h

Hold: 8000 V, 11 h

4. Inter-dimension Process

4.1 Protocol

4.1.1 Step name

equilibration

4.1.2 Inter dimension buffer

first SDS-PAGE equilibration buffer :
50 mM Tris-HCl (pH 8.8)
6 M urea
30 % (v/v) glycerol
2 % (w/v) SDS
0.002 % (w/v) bromophenol blue

4.1.3 Additional reagents

60 mM DTT

4.1.4 Equipment

4.1.5 Protocol

Temperature: 20 °C.

Duration: 30 min.

4.1.1 Step name

equilibration

4.1.2 Inter dimension buffer

second SDS-PAGE equilibration buffer :
50 mM Tris-HCl (pH 8.8)
6 M urea
30 % (v/v) glycerol
2 % (w/v) SDS
0.002 % (w/v) bromophenol blue

4.1.3 Additional reagents

130 mM iodoacetamide

4.1.4 Equipment

4.1.5 Protocol

Temperature: 20 °C.

Duration: 15 min.

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:

12 % T acrylamide (29:1 acrylamide-bisacrylamide
ratio) gel
375 mM Tris-HCl (pH 8.8)
0.1 % SDS
0.005 % (w/v) ammonium persulfate
0.05 % (v/v) TEMED

3.2.3 Physical dimensions

X: 255 mm
Y: 205 mm
Z: 1 mm

3.2.4 Physiochemical property range and distribution

logarithmic apparent molecular mass 100 - 10 kDa

3.2.5 Acrylamide concentration

12 %

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 29:1

3.2.7 Additional substances in gel

No additional substance

3.2.8 Gel lane

1

3.2.9 Sample application

Loading method: strip application.
Additional comment: The strips were sealed to the gel surface with a 60°C
solution of 375 mM Tris-HCl (pH 8.8), 0.1 % (w/v) SDS, 0.002 % (w/v) bromophenol blue, and 0.5 % (w/v) low melting point agarose

3.3 Protocol

3.3.1 Buffers

25 mM Tris-HCl (pH 8.3)
192 mM glycine
0.1 % SDS (w/v)

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Hold: 15 mA, 16 h

5. Detection

5.1 Direct detection

5.1.1 Name of direct detection_process

colloidal Coomassie Blue staining

5.1.2 Direct detection agents

Coomassie Serva Blue G-250

5.1.3 Additional reagents and buffers

No additional reagents or buffer

5.1.4 Equipment

Manufacturer: BIO-RAD
Model: Dodeca Stainer
Model number: 165-3400

5.1.5 Direct detection protocol

Temperature: 20 °C.

Duration: 3 d.

Protocol:
After migration, gel was fixed for 3 h in 50 % (v/v) ethanol/2 % (v/v) phosphoric acid, washed for 3 x 30 min in Milli-Q water, and incubated for 1 h in 34 % (v/v) methanol/17 % (w/v) ammonium sulfate/3 % (v/v) phosphoric acid. Coomassie Serva Blue G-250 powder was added to a concentration of 700 mg/l and the gels stained for 3 days and destained with Milli-Q water.

Detection is described in the following reference protocol:
Citation: Electrophoresis,6, page(s) 427-448 (1985).
URL: not provided.

6. Image Acquisition

6.1 Acquisition Equipment

6.1.1 Type of equipment

scanner

6.1.2 Name of equipment

Manufacturer: GE Healthcare
Model: ImageScanner
Model number: 18-1170-84

6.1.3 Software

Manufacturer: GE Healthcare
Model: LabScan software
Model number: 5.0

6.1.4 Calibration

No (manual)

6.1.5 Equipment specific parameters

Default (vendor) parameters.

6.2 Acquisition Protocol

6.2.1 Image acquisition process

Gel images were acquired on an ImageScanner (GE Healthcare Life Sciences) using LabScan 5 software at 300 dpi and were saved as TIFF and mel files

6.2.2 Reference to gel matrix

There is only one gel in this document.

7. Image

7.1.1 Image name (or id)

gel448_BY2_ext_chloromethanol_1mg_A2.tiff (format: TIFF)

7.1.2 Dimensions

Width: 2982 px

Height: 2454 px

7.1.3 Resolution

300 dpi

7.1.4 Bit-depth

16-bit (HighColor)

7.1.5 Image location

FigonlineBIG.jpg

7.1.6 Standard image orientation

Yes

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