Name: C. elegans proteome after infection with Staphylococcus aureus Description: C. elegans proteome after infection with the gram-negative bacterium Aeromonas hydrophila Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2009-09-16 1.1.2 Responsible person or role Affiliation: Katholieke Universiteit Leuven (i) Name: Prof. Dr. Liliane Schoofs (ii) Postal address: Department of Biology Research group of Functional Genomics and Proteomics Naamsestraat 59 3000 Leuven Belgium (iii) Email address: Liliane.Schoofs@bio.kuleuven.be 1.1.3 Electrophoresis type Two-Dimensional electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: proteome of C. elegans challenged with S. aureus 2. Sample type: Control sample 2.1.2 Loading buffer 1. Urea (7 M) Thiourea (2 M) 4% CHAPS 40 mM Tris 1% DDT 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 Destreak rehydration solution (GE Healthcare, 17-6003-19) 3.2.8 Gel lane 1 3.2.9 Sample application Lane 1 * Sample: proteome of C. elegans challenged with S. aureus * Volume of sample: 300 µg * Loading buffer: Urea (7 M) Thiourea (2 M) 4% CHAPS 40 mM Tris 1% DDT * Volume of loading buffer: 50 µL Loading method: cup loading. 3.3 Protocol 3.3.1 Buffers No buffer. 3.3.2 Electrophoresis conditions Running temperature: 20 °C Hold: 150 V, 3 h Hold: 300 V, 3 h Hold: 1000 V, 6 h Hold: 8000 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 in the first step and 4% (w/v) iodoacetamide Buffer B: buffer A in which DTT was replaced with 4% (w/v) iodoacetamide 4.1.3 Additional reagents A trace of bromophenol blue in the second equilibration step 4.1.4 Equipment Manufacturer: GE Healthcare Bio-Sciences Corp. Model: Immobiline Drystrip Reswelling Tray Model number: 80-6465-32 4.1.5 Protocol Temperature: 20 °C. Duration: 30 min. Protocol: 15 min in buffer A followed by 15 min 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: 30 % Acrylamide/Bis (250 ml), Tris-Cl pH 8.8 (150 ml), Double distilled water (187 ml), 10% SDS (6 ml), 10% APS (6 ml), 10% TEMED (830 µl) 3.2.3 Physical dimensions X: 260 mm Y: 200 mm Z: 1 mm 3.2.4 Physiochemical property range and distribution logarithmic apparent molecular mass 10 - 200 kDa 3.2.5 Acrylamide concentration 12.48 % 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: Bisacrylamide Ratio: 37.5:1 3.2.7 Additional substances in gel No additional substances 3.2.8 Gel lane 1 3.2.9 Sample application Loading method: IPG transfer. 3.3 Protocol 3.3.1 Buffers 3 X SDS 1 X SDS 3.3.2 Electrophoresis conditions Running temperature: 20 °C Hold: 8 mA, 1 h Hold: 12 mA, 12 h 5. Detection ------------ 5.1 Direct detection 5.1.1 Name of direct detection_process Silver staining 5.1.2 Direct detection agents 0.1 % silver nitrate 5.1.3 Additional reagents and buffers 1. 50% methanol and 5% acetic acid 2. 50% methanol 3. milli-Q 4. 0.02% sodium thiosulfate 5. 2% sodium carbonate and 0,04% formaldehyde 6. 5% acetic acid 7. 1% acetic acid 5.1.4 Equipment Manufacturer: unknown Model: no specialised equipment Model number: unknown 5.1.5 Direct detection protocol Temperature: 20 °C. Duration: 1.5 h. Detection is described in the following reference protocol: Citation: Anal. Chem.,68, page(s) 850-858 (1996). URL: not provided. 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment camera 6.1.2 Name of equipment Manufacturer: Nikon Model: Nikon Coolpix 990 Model number: unknown 6.1.3 Software Manufacturer: unknown Model: no software used Model number: unknown 6.1.4 Calibration Yes (automatic) 6.1.5 Equipment specific parameters unknown 6.2 Acquisition Protocol 6.2.1 Image acquisition process UNKNOWN 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) Ce_Sa_silver (format: TIFF) 7.1.2 Dimensions Width: 1000 px Height: 1000 px 7.1.3 Resolution 500 dpi 7.1.4 Bit-depth 8-bit (256 colors) 7.1.5 Image location C:\Documents and Settings\Annelies\Desktop\CE-SA paper\grijs_zonder_tekst-1000x1000.tif Link: /download/22/6Nhvx7OY/ 7.1.6 Standard image orientation Yes