Name: Pseudomonas putida UW4 proteome reference map
Description: Construction of the proteome reference map of a plant growth-promoting bacterium Pseudomonas putida UW4 using 2-D gel and mass spectrometry.
Version: MIAPE: Gel Electrophoresis 1.4
1. General features
1.1.1 Date Stamp
2007-02-27
1.1.2 Responsible person or role
Affiliation: experimenter
(i) Name: Zhenyu Cheng
(ii) Postal address: 200 University Avenue West
Waterloo, Ontario
N2L 3V2
Canada
(iii) Email address: czhenyu@uwaterloo.ca
1.1.3 Electrophoresis type
Two-Dimensional electrophoresis
2. Sample
2.1.1 Sample Name(s)
-
- Sample name: Pseudomonas putida UW4 isolated from rhisosphere
- Sample type: Standard
2.1.2 Loading buffer
- 2 M thiourea, 7 M urea, 4% w/v CHAPS, 30 mM Tris pH 8.5, 60 mM DTT, 0.5% v/v IPG pH 4-7 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: GE Healthcare
Model: Immobiline DryStrip
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: 1:3
3.2.7 Additional substances in gel
No additional substance
3.2.8 Gel lane
13.2.9 Sample application
Lane 1
- Sample: Pseudomonas putida UW4 isolated from rhisosphere
- Volume of sample: 1 mg
- Loading buffer: 2 M thiourea, 7 M urea, 4% w/v CHAPS, 30 mM Tris pH 8.5, 60 mM DTT, 0.5% v/v IPG pH 4-7 buffer, and a trace of bromophenol blue
- Volume of loading buffer: 450 µL
Loading method: rehydration loading.
3.3 Protocol
3.3.1 Buffers
2 M thiourea, 7 M urea, 4% w/v CHAPS, 30 mM Tris pH 8.5, 60 mM DTT, 0.5% v/v IPG pH 4-7 buffer, and a trace of bromophenol blue
3.3.2 Electrophoresis conditions
Running temperature: 20 °C
Hold: 500 V, 1 h
Hold: 500.1000 V, 3 h
Hold: 1000.3000 V, 3 h
Hold: 3000 V, 2 h
Hold: 3000.8000 V, 3 h
Hold: 8000 V, 10.5 h
4. Inter-dimension Process
4.1 Protocol
4.1.1 Step name
equilibration
4.1.2 Inter dimension buffer
6M Urea, 2% w/v SDS, 50mM Tris-HCl pH 8.8, 30% v/v Glycerol, 0.002% (w/v) bromophenol blue)
4.1.3 Additional reagents
DTT (10mg/ml)
4.1.4 Equipment
Manufacturer: VWR
Model: rocking platform
Model number: 100
4.1.5 Protocol
Temperature: 20 °C.
Duration: 20 min.
Protocol:
shaking for 20 minutes
4.1.1 Step name
equilibration
4.1.2 Inter dimension buffer
6M Urea, 2% w/v SDS, 50mM Tris-HCl pH 8.8, 30% v/v Glycerol, 0.002% (w/v) bromophenol blue)
4.1.3 Additional reagents
25mg/ml iodoacetamide
4.1.4 Equipment
Manufacturer: VWR
Model: rocking platform
Model number: 100
4.1.5 Protocol
Temperature: 20 °C.
Duration: 20 min.
Protocol:
shaking for 20 minutes
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:
H2O 33 mL, 30%acrylamide mix 40ml, 1.5M Tris (pH8.8) 25ml, 10% SDS 1ml, 10% ammonium persulfate 1ml, and 0.04ml of TEMED
3.2.3 Physical dimensions
X: 24 cm
Y: 18 cm
Z: 0.1 cm
3.2.4 Physiochemical property range and distribution
logarithmic apparent molecular mass 150 - 5 kDa
3.2.5 Acrylamide concentration
12 %
3.2.6 Acrylamide : Crosslinker ratio
Crosslinker: Bisacrylamide
Ratio: 37.5:1
3.2.7 Additional substances in gel
No additional substance
3.2.8 Gel lane
13.2.9 Sample application
Loading method: well loading.
3.3 Protocol
3.3.1 Buffers
25 mM Tris-HCl pH8.3, 192 mM glycine, 0.1%SDS
3.3.2 Electrophoresis conditions
Running temperature: 10 °C
Hold: 17.5 mA, 6 h
5. Detection
5.1 Direct detection
5.1.1 Name of direct detection_process
Coomassie blue staining
5.1.2 Direct detection agents
1L of 2 g/L coomassie G-250, 1L of 1M sulfuric acid, 220 mL of 10M sodium hydroxide, 310 mL of 100% TCA
5.1.3 Additional reagents and buffers
No additional reagents or buffer
5.1.4 Equipment
Manufacturer: VWR
Model: rocking platform
Model number: 100
5.1.5 Direct detection protocol
Temperature: 20 °C.
Duration: 18 h.
Protocol:
shaking overnight
6. Image Acquisition
6.1 Acquisition Equipment
6.1.1 Type of equipment
fluorescent scanner6.1.2 Name of equipment
Manufacturer: GE Healthcare
Model: Typhoon
Model number: 9400
6.1.3 Software
Manufacturer: GE Healthcare
Model: Typhoon scanner control
Model number: 3.0
6.1.4 Calibration
Yes (automatic)
6.1.5 Equipment specific parameters
Default (vendor) parameters.6.2 Acquisition Protocol
6.2.1 Image acquisition process
excitation wavelength of 633nm and without an emission filterPMT 600
scan time 15 min
6.2.2 Reference to gel matrix
There is only one gel in this document.
7. Image
7.1.1 Image name (or id)
Figure 1 (format: TIFF)
7.1.2 Dimensions
Width: 2294 px
Height: 1845 px
7.1.3 Resolution
254 dpi
7.1.4 Bit-depth
8-bit (256 colors)7.1.5 Image location
Cheng_database.tif7.1.6 Standard image orientation
Yes