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. 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 Link: /download/318/veNnKjKh/ 7.1.6 Standard image orientation Yes