Name: Protochlamydia amoebophila UWE25 elementary body reference proteome map

Description: Construction of a proteome reference map of the infectious stage (the elementary bodies) of Protochlamydia amoebophila UWE25, an endosymbiont of Acanthamoeba spp., using 2-D gel electrophoresis and mass spectrometric protein identification.

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2005-10-01

1.1.2 Responsible person or role

Affiliation: University of Vienna, Department of Microbial Ecology

(i) Name: Matthias Horn

(ii) Postal address: University of Vienna
Department of Microbial Ecology
Althanstrasse 14
1090 Vienna
Austria

(iii) Email address: horn@microbial-ecology.net

1.1.3 Electrophoresis type

Two-Dimensional electrophoresis

2. Sample

2.1.1 Sample Name(s)

    1. Sample name: Proteins extracted from purified P. amoebophila elementary bodies
    2. Sample type: Standard

2.1.2 Loading buffer

  1. 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 40 mM Tris, 1% (w/v) DTT, 2% (v/v) IPG buffer pH 3-10 (GE Healthcare Bio-Sciences Corp.), 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 Bio-Sciences Corp
Model: ImmobilineTM DryStrip pH 3-10 Non-linear 24 cm strips
Model number: 17-6002-45
Batch number: unknown

3.2.3 Physical dimensions

X: 240 mm
Y: 3 mm
Z: 0.5 mm

3.2.4 Physiochemical property range and distribution

sigmoidal pH 3 - 10

3.2.5 Acrylamide concentration

4 %

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 32:1

3.2.7 Additional substances in gel

none

3.2.8 Gel lane

1

3.2.9 Sample application

Lane 1

  • Sample: Proteins extracted from purified P. amoebophila elementary bodies
  • Volume of sample: 500 µg
  • Loading buffer: 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 40 mM Tris, 1% (w/v) DTT, 2% (v/v) IPG buffer pH 3-10 (GE Healthcare Bio-Sciences Corp.), and a trace of bromophenol blue
  • Volume of loading buffer: 450 µL

Loading method: rehydration loading.
Additional comment: Passive rehydration

3.3 Protocol

3.3.1 Buffers

No buffer.

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Hold: 500 V, 1 h

Hold: 2000 V, 1 h

Hold: 10000 V, 6 h

4. Inter-dimension Process

4.1 Protocol

4.1.1 Step name

equilibration

4.1.2 Inter dimension buffer

Buffer A: 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 50 mM Tris-HCl (pH 8.8) and 1% (w/v) DTT
Buffer B: buffer A in which DTT was replaced with 4% (w/v) iodoacetamide

4.1.3 Additional reagents

No additional reagent.

4.1.4 Equipment

4.1.5 Protocol

Duration: 30 min.

Protocol:
15 min incubation in buffer A, followed by 15 min incubation in buffer B

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:

amount for six gels: 30 % (w/v) Acrylamide/Bisacrylamide (188 ml), 1.5 M Tris-Cl pH 8.8 (113 ml), Double distilled water (140 ml), 10% (w/v) SDS (4.5 ml), 10% (w/v) ammonium persulfate (4.5 ml), 10% (w/v) TEMED (620 µl)

3.2.3 Physical dimensions

X: 240 mm
Y: 205 mm
Z: 1.5 mm

3.2.4 Physiochemical property range and distribution

logarithmic apparent molecular mass 10 - 120 kDa

3.2.5 Acrylamide concentration

12.5 %

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 37.5:1

3.2.7 Additional substances in gel

none

3.2.8 Gel lane

1

3.2.9 Sample application

Loading method: IPG transfer.

3.3 Protocol

3.3.1 Buffers

cathode buffer: 3 X SDS electrophoresis buffer (75 mM Tris, 576 mM Glycine, 0.3% (w/v) SDS)

anode buffer: 1 X SDS electrophoresis buffer (25 mM Tris, 192 mM Glycine, 0.1% (w/v) SDS)

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Hold: 0 V, 30 min

Hold: 0 V, 3.5 h

5. Detection

5.1 Direct detection

5.1.1 Name of direct detection_process

Colloidal Coomassie blue staining

5.1.2 Direct detection agents

0.6 g/L Coomassie brillant blue G-250, 100 g/L ammonium sulfate, 2% phosphoric acid, 25% Methanol

5.1.3 Additional reagents and buffers

Fixation solution: 40% ethanol, 10% acetic acid

5.1.4 Equipment

No specialised equipment.

5.1.5 Direct detection protocol

Duration: 1 d.

Protocol:
The gel was fixed for 30 min in fixation solution, washed with distilled water and stained overnight in Colloidal Coomassie staining solution.

6. Image Acquisition

6.1 Acquisition Equipment

6.1.1 Type of equipment

flatbed color scanner

6.1.2 Name of equipment

Manufacturer: Seiko Epson Corp.
Model: Epson Expression 1680 Pro
Model number: unknown

6.1.3 Software

Manufacturer: LaserSoft Imaging GmbH
Model: SilverFast® Ai 6
Model number: Photoshop Version

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

The gel image was acquired in 16 bit grayscale with a resolution of 300 dpi and was saved in tif format. No filters or image processing tools were used during aquisition. A Kodak Step Tablet no. 2 (GE Healthcare Bio-Sciences Corp.) was scanned using the same parameters.
An intensity calibration was performed within the image analysis software Image Master 2D Platinum (GE Healthcare Bio-Sciences Corp.) as described in the manual of the software.

6.2.2 Reference to gel matrix

There is only one gel in this document.

7. Image

7.1.1 Image name (or id)

Pam EB gel (format: TIFF)

7.1.2 Dimensions

Width: 768 px

Height: 1040 px

7.1.3 Resolution

72 dpi

7.1.4 Bit-depth

24-bit (TrueColor)

7.1.5 Image location

Protochlamydia amoebophila reference proteome map.tif

7.1.6 Standard image orientation

Yes

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