Name: C. elegans - S. aureus 4 hours Description: The C. elegans proteome 4 hours after infection with S. aureus 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 Difference gel electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: 75µg of naive C. elegans proteins labeled with Cy3, 75µg of immune-challenged C. elegans proteins labeled with Cy5, 75 µg of internal standard labeled with Cy2 2. Sample type: Test sample 2. 1. Sample name: 75µg of naive C. elegans proteins labeled with Cy5, 75µg of immune-challenged C. elegans proteins labeled with Cy3, 75 µg of internal standard labeled with Cy2 2. Sample type: Test sample 3. 1. Sample name: 75µg of naive C. elegans proteins labeled with Cy3, 75µg of immune-challenged C. elegans proteins labeled with Cy5, 75 µg of internal standard labeled with Cy2 2. Sample type: Test sample 4. 1. Sample name: 75µg of naive C. elegans proteins labeled with Cy5, 75µg of immune-challenged C. elegans proteins labeled with Cy3, 75 µg of internal standard labeled with Cy2 2. Sample type: Test sample 5. 1. Sample name: 75µg of naive C. elegans proteins labeled with Cy3, 75µg of immune-challenged C. elegans proteins labeled with Cy5, 75 µg of internal standard labeled with Cy2 2. Sample type: Test sample 6. 1. Sample name: 75µg of naive C. elegans proteins labeled with Cy5, 75µg of immune-challenged C. elegans proteins labeled with Cy3, 75 µg of internal standard labeled with Cy2 2. Sample type: Test 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: 75µg of naive C. elegans proteins labeled with Cy3, 75µg of immune-challenged C. elegans proteins labeled with Cy5, 75 µg of internal standard labeled with Cy2 * Volume of sample: 225 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 Fluorescent staining 5.1.2 Direct detection agents Cy dyes: Cy3, Cy5, Cy2 (GE Healtcare: 5 nmol CyDye DIGE Fluor (minimal Dye) Labelling Kit), 0.267 nmol per Cy Dye per sample (see 2.1.1) 5.1.3 Additional reagents and buffers 100% DMF, 0,128% (w/v) lysine 5.1.4 Equipment Manufacturer: unknown Model: no specialised equipment Model number: unknown 5.1.5 Direct detection protocol Temperature: 20 °C. Duration: 1 h. Protocol: Cy Dye is solved in 12,5 µl of 100% DMF, 2 µl of the Cy3 or the Cy5 Dye solution is added separatly to 75 µg protein of each test and each control sample. 12 µl of the Cy2 Due solution is added to the internal standard protein mixture, which contains 75 µg protein from each sample. Minimal labeling is done for 45 min at 4°C in the dark. Then, 1 µl of the lysine solution added to each sample to stop the labeling reaction (10 min 4°C in the dark). 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment fluorescent scanner 6.1.2 Name of equipment Manufacturer: GE Healthcare Bio-Sciences Corp. Model: Ettan DIGE Imager Model number: unknown 6.1.3 Software Manufacturer: GE Healthcare Bio-Sciences Corp. Model: Ettan TM DIGE Imager Software Model number: unknown 6.1.4 Calibration Yes (automatic) 6.1.5 Equipment specific parameters Cy3 excitation filter 540/25 nm and emission filter 595/25 nm Cy5 excitation filter 635/30 nm and emission filter 680/25 nm Cy2 excitation filter 480/30 nm and emission filter 530/40 nm 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_4h (format: BMP) 7.1.2 Dimensions Width: 490 px Height: 465 px 7.1.3 Resolution 96 dpi 7.1.4 Bit-depth 32-bit (TrueColor) 7.1.5 Image location C:\Documents and Settings\Annelies\Desktop\Ce_Sa_4h.bmp Link: /download/22/ZWRio4Ao/ 7.1.6 Standard image orientation Yes