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Name: Proteome reference map for Xanthomonas oryzae pv. oryzae ZJ173 (pH 4-7)

Description: Construction of the proteome reference map(pH 4-7) of the plant-pathogenic bacterium Xanthomonas oryzae pv. oryzae ZJ173 using 2-D gel and MALDI-TOF-TOF MS.

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2011-12-10

1.1.2 Responsible person or role

Affiliation: experimenter

(i) Name: Shu Xu

(ii) Postal address: College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China

(iii) Email address: 2009202039@njau.edu.cn

1.1.3 Electrophoresis type

Two-Dimensional electrophoresis

2. Sample

2.1.1 Sample Name(s)

    1. Sample name: Xanthomonas oryzae pv. oryzae
    2. Sample type: Standard

2.1.2 Loading buffer

  1. 8 M urea, 2 M thiourea, 4% CHAPS (w/v), 0.2% (w/v) Bio-Lyte (pH 3-10), 0.001% (w/v) bromophenol blue, 1 mM PMSF, and 2 mM EDTA

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: IPG strip 17 cm; pH 4-7
Model number: 163-2008
Batch number: unknown

3.2.3 Physical dimensions

X: 17.8 cm
Y: 0.33 cm
Z: 0.05 cm

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: Xanthomonas oryzae pv. oryzae
  • Volume of sample: 800 µg
  • Loading buffer: 8 M urea, 2 M thiourea, 4% CHAPS (w/v), 0.2% (w/v) Bio-Lyte (pH 3-10), 0.001% (w/v) bromophenol blue, 1 mM PMSF, and 2 mM EDTA
  • Volume of loading buffer: 350 µL

Loading method: rehydration loading.

3.3 Protocol

3.3.1 Buffers

No buffer.

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Gradient: 0-100 V, 0.5 h

Gradient: 100-250 V, 0.5 h

Gradient: 250-500 V, 0.5 h

Hold: 1000 V, 1 h

Gradient: 1000-10000 V, 5 h

Hold: 10000 V, 3.5 h

4. Inter-dimension Process

4.1 Protocol

4.1.1 Step name

reduction

4.1.2 Inter dimension buffer

6 M urea, 2% SDS, 20% (v/v) glycerol, 0.375 M Tris-HCl pH 8.8, and 20mg/ml DTT

4.1.3 Additional reagents

No additional reagent.

4.1.4 Equipment

Manufacturer: Nanjing University
Model: rocking platform
Model number: unknown

4.1.5 Protocol

Temperature: 20 °C.

Duration: 15 min.

Protocol:
shaking for 15 min

4.1.1 Step name

alkylation

4.1.2 Inter dimension buffer

6M urea, 2% SDS, 20% (v/v) glycerol, 0.375 M Tris-HCl pH 8.8, and 25mg/ml iodoacetamide

4.1.3 Additional reagents

No additional reagent.

4.1.4 Equipment

Manufacturer: Nanjing University
Model: rocking platform
Model number: unknown

4.1.5 Protocol

Temperature: 20 °C.

Duration: 15 min.

Protocol:
shaking for 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:

For each independent experiment, six gels were manufactured using the following receipe:
161.2 ml Milli-Q water, 124.8 ml 30 % acrylamide/Bis solution, 98.8 ml buffer (1.5 M Tris-HCl pH 8.8, 4% SDS), 5.8 ml 10% APS solution, 260 µl TEMED

3.2.3 Physical dimensions

X: 24 cm
Y: 18 cm
Z: 0.1 cm

3.2.4 Physiochemical property range and distribution

linear apparent molecular mass 150 - 20 kDa

3.2.5 Acrylamide concentration

10 %

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: IPG strip placing.

3.3 Protocol

3.3.1 Buffers

25 mM Tris-HCl pH 8.3, 192 mM glycine, 0.1 % SDS

3.3.2 Electrophoresis conditions

Running temperature: 10 °C

Hold: 60 mA, 0.25 h

Hold: 120 mA, 5 h

5. Detection

5.1 Direct detection

5.1.1 Name of direct detection_process

Coomassie blue staining

5.1.2 Direct detection agents

Fixation solution(2L): 40% methanol, 10% acetic acid
Staining solution(2L): Coomassie G-250 2.4 g, phosphoric acid 232ml, ammonium sulfate 200g , methanol 400 ml

5.1.3 Additional reagents and buffers

No additional reagents or buffer

5.1.4 Equipment

Manufacturer: BioRad
Model: GS-800 scanner
Model number: 170-7981

5.1.5 Direct detection protocol

Detection is described in the following reference protocol:
Citation: Electrophoresis,25, page(s) 1327-1333 (2004).
URL: not provided.

6. Image Acquisition

6.1 Acquisition Equipment

6.1.1 Type of equipment

Calibrated Densitometer

6.1.2 Name of equipment

Manufacturer: BioRad
Model: GS-800 scanner
Model number: 170-7981

6.1.3 Software

Manufacturer: BioRad
Model: Quantity One
Model number: 4.62

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

'gel' and 'Coomassie Blue' were chosen, and image acquisition was conducted after preview scan.

6.2.2 Reference to gel matrix

There is only one gel in this document.

7. Image

7.1.1 Image name (or id)

4-7 (format: TIFF)

7.1.2 Dimensions

Width: 2205 px

Height: 2223 px

7.1.3 Resolution

400 dpi

7.1.4 Bit-depth

8-bit (256 colors)

7.1.5 Image location

C:\\\\Users\\\\Administrator\\\\Desktop\\\\4-7.tif

7.1.6 Standard image orientation

Yes

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