Name: 2-DE and 2-DE Western blotting of OMPs of B. melitensis M5

Description: 2-DE and 2-DE Western blotting of OMPs of B. melitensis M5

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2009-08-31

1.1.2 Responsible person or role

Affiliation: Department of Immunology

(i) Name: Beijing Institute of Microbiology & Epidemiology

(ii) Postal address: No.20 Dongda street, Fengtai District, Beijing 100071

(iii) Email address: xiaozzp@eyou.com

1.1.3 Electrophoresis type

Two-Dimensional electrophoresis

2. Sample

2.1.1 Sample Name(s)

    1. Sample name: Soluble proteins of M5
    2. Sample type: Test sample

2.1.2 Loading buffer

  1. 8M urea, 1% DTT, 4% CHAPS, suitable protease inhibitor

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: Amersham pharmaci
Model: IPG 4.0-7.0
Model number: 17-1233-01
Batch number: Amersham pharmaci 20043079

3.2.3 Physical dimensions

X:
Y:
Z:

3.2.4 Physiochemical property range and distribution

-

3.2.5 Acrylamide concentration

%

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker:
Ratio: :

3.2.7 Additional substances in gel

No additional substance

3.2.8 Gel lane

1

3.2.9 Sample application

Lane 1

  • Sample: Soluble proteins of M5
  • Volume of sample: 1 mg
  • Loading buffer: 8M urea, 1% DTT, 4% CHAPS, suitable protease inhibitor
  • 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

Hold: 30 V, 10.00 h

Hold: 500 V, 30 min

Hold: 2000 V, 30 min

Hold: 5000 V, 30 min

Gradient: 5000-10000 V, 2 h

Hold: 10000 V, 8 h

4. Inter-dimension Process

4.1 Protocol

4.1.1 Step name

reduction

4.1.2 Inter dimension buffer

6 M urea,30% glycerol,1%DTT

4.1.3 Additional reagents

No additional reagent.

4.1.4 Equipment

4.1.5 Protocol

Temperature: 20 °C.

Duration: 15 min.

4.1.1 Step name

alkylation

4.1.2 Inter dimension buffer

0.05 M Tris -HCl , pH 8.8 ,6 M urea, 30% (w/v) glycerol
and 2% (w/v) SDS,4%iodoacetamide.

4.1.3 Additional reagents

No additional reagent.

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 reference protocol:

Citation: Chinese J ourna1 of Zoonoses,23(12), page(s) 1172-1175 (2007).
URL: http://www.cqvip.com

3.2.3 Physical dimensions

X:
Y:
Z:

3.2.4 Physiochemical property range and distribution

-

3.2.5 Acrylamide concentration

%

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker:
Ratio: :

3.2.7 Additional substances in gel

No additional substance

3.2.8 Gel lane

1

3.2.9 Sample application

Loading method: paper bridge loading.

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: 20 °C

Hold: 20 mA, 45 min

Hold: 30 mA, 4 h

5. Detection

5.2 Indirect detection

5.2.1 Name of direct detection_process

N/A

5.2.2 Transfer medium

N/A

Manufacturer: N/A
Model: N/A
Model number: N/A

5.2.3 Detection medium

N/A

Manufacturer: N/A
Model: N/A
Model number: N/A

5.2.4 Indirect detection agents

N/A

5.2.5 Additional reagents and buffers

No additional reagents or buffer

5.2.6 Equipment

No specialised equipment.

5.2.7 Indirect detection protocol

6. Image Acquisition

6.1 Acquisition Equipment

6.1.1 Type of equipment

laser scanner

6.1.2 Name of equipment

Manufacturer: Amersham pharmaci
Model: Imagescanner
Model number: 18-1134-45

6.1.3 Software

Manufacturer: Amersham pharmaci
Model: ImageMaster TM 2D platinum
Model number: version5

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

Western blotting was performed according to the method of Towbin et al. Proteins were transferred to PVDF membranes using TE77 ECL semi-dry transfer unit (0.8 mA/cm2, 1h). After completion of transfer, non-specific binding sites on the membranes were blocked for 90 min with 5% skimmed milk in TBS at 37℃. Then, PVDF membranes were incubated with primary antibody, rabbit anti-B. melitensis M5 (the agglutination titer of the pooled sera was 1:3200), diluted 1:50 in TBS containing 5% skimmed milk for 1 h at 37℃ on a gentle shaker. The membranes were rinsed in 0.1% Tween20 TBS three times, 10 min each, and incubated with goat anti-rabbit-HRP at a dilution of 1:10000 in TBS containing 5% skimmed milk for 1 h at 37℃. After washing, the blots were developed using ECL Western blotting detection reagents. The specific immunogenic protein pattern was visualized on an X-ray film.

6.2.2 Reference to gel matrix

There is only one gel in this document.

7. Image

7.1.1 Image name (or id)

omp (format: TIFF)

7.1.2 Dimensions

Width: 1000 px

Height: 1000 px

7.1.3 Resolution

400 dpi

7.1.4 Bit-depth

32-bit (TrueColor)

7.1.5 Image location

C:\Documents and Settings\immun323.AMMS-49CC6649A4\桌面\OMP.doc

7.1.6 Standard image orientation

Yes

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