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)

    1. Sample name: Pseudomonas putida UW4 isolated from rhisosphere
    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, 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

1

3.2.9 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

1

3.2.9 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 scanner

6.1.2 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 filter
PMT 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.tif

7.1.6 Standard image orientation

Yes

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