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)
-
- Sample name: Proteins extracted from purified P. amoebophila elementary bodies
- Sample type: Standard
2.1.2 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
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
13.2.9 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
13.2.9 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 scanner6.1.2 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.tif7.1.6 Standard image orientation
Yes