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Name: Proteomes of laboratory-grown B.melitensis strains M5

Description: Proteomes of laboratory-grown B.melitensis strains M5 in the pH 4.0 to 7.0.

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2009-08-30

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 20043077

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.
Additional comment: Procedures for 2-DE were carried out according to the manufacturer’s instructions. The sample proteins (1 mg) were separated by isoelectric focusing (IEF) on 18 cm, pH 4-7 linear immobilized pH gradient (IPG) strips. After 12 h of rehydration with 30 V at 20℃

3.3 Protocol

3.3.1 Buffers

No buffer.

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

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

5.1 Direct detection

5.1.1 Name of direct detection_process

Coomassie blue staining

5.1.2 Direct detection agents

Coomassie blue staining R35O 1 tablet,1600ml 10% Acetic Acid

5.1.3 Additional reagents and buffers

No additional reagents or buffer

5.1.4 Equipment

No specialised equipment.

5.1.5 Direct detection protocol

Temperature: 100 °C.

Duration: 10 min.

Detection is described in the following reference protocol:
Citation: Chinese J ourna1 of Zoonoses,23(12), page(s) 1172-1175 (2007).
URL: http://www.cqvip.com

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

400dpi

6.2.2 Reference to gel matrix

There is only one gel in this document.

7. Image

7.1.1 Image name (or id)

M5 (format: TIFF)

7.1.2 Dimensions

Width: 10000 px

Height: 10000 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\M5.tif

7.1.6 Standard image orientation

Yes

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