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)
-
- Sample name: 1 mg of soluble proteins from Nicotiana tabacum Bright- Yellow-2 BY2 cells
- Sample type: Test sample
2.1.2 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
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
13.2.9 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
13.2.9 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
scanner6.1.2 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 files6.2.2 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.jpg7.1.6 Standard image orientation
Yes