Name: FNSinip310g63347 Description: Insoluble fraction: comparison of C. elegans proteins from worms infected or not by the fungus Drechmeria coniospora Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2008-07-15 1.1.2 Responsible person or role Affiliation: Centre d'Immunologie de Marseille Luminy (i) Name: Dr Jonathan Ewbank (ii) Postal address: Research group of Innate immunity in C. elegans Case 906 163 avenue de luminy 13288 Marseille Cedex 9 France (iii) Email address: ewbank@ciml.univ-mrs.fr 1.1.3 Electrophoresis type Two-Dimensional electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: 50 µg of infected C. elegans proteins Cy3 labeled , 50 µg of naive C. elegans proteins Cy5 labeled , 50 µg internal standard Cy2 labeled 2. Sample type: Test sample 2. 1. Sample name: 50 µg of naive C. elegans proteins Cy3 labeled , 50 µg of infected C. elegans proteins Cy5 labeled , 50 µg internal standard Cy2 labeled 2. Sample type: Test sample 2.1.2 Loading buffer 1. Urea 7M Thiourea 2M CHAPS 4% Ampholytes 1% 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-healtcare Model: ImmobilineTM DryStrip pH 3-10 linear 11 cm strips Model number: 18-1016-61 Batch number: 63341-63347 3.2.3 Physical dimensions X: 108 mm Y: 3 mm Z: 0.5 mm 3.2.4 Physiochemical property range and distribution linear 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: ref 17-6003-19 GE Healthcare 3.2.8 Gel lane 1 3.2.9 Sample application Lane 1 * Sample: 50 µg of infected C. elegans proteins Cy3 labeled , 50 µg of naive C. elegans proteins Cy5 labeled , 50 µg internal standard Cy2 labeled * Volume of sample: 150 µg * Loading buffer: Urea 7M Thiourea 2M CHAPS 4% Ampholytes 1% * Volume of loading buffer: 35 µL Loading method: cup loading. 3.3 Protocol 3.3.1 Buffers No buffer. 3.3.2 Electrophoresis conditions Running temperature: 20 °C Gradient: 0-30 V, 3 h Gradient: 30-300 V, 3 h Gradient: 300-6000 V, 8 h Hold: 6000 V, 6 h 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name equilibration 4.1.2 Inter dimension buffer 6 M Urea; 50 mM Tris-HCl pH 8,8; 34.5 %(v/v)Glycerol; 2 %(v/v)SDS; 65mM DTT; trace of Bromophenol blue 4.1.3 Additional reagents No additional reagent. 4.1.4 Equipment 4.1.5 Protocol Temperature: 20 °C. Duration: 20 min. Protocol: 20 minutes in equilibration buffer 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 purchased precast. Manufacturer: Bio-rad Model: criterion XT bis-tris gel 10% Model number: 3450115 Batch number: unknown 3.2.3 Physical dimensions X: 133 mm Y: 87 mm Z: 1 mm 3.2.4 Physiochemical property range and distribution logarithmic apparent molecular mass 10 - 250 kDa 3.2.5 Acrylamide concentration 10 % 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: Bisacrylamide Ratio: 10:5 3.2.7 Additional substances in gel No additional substance 3.2.8 Gel lane 1 3.2.9 Sample application Loading method: well loading. 3.3 Protocol 3.3.1 Buffers MOPS 1x 3.3.2 Electrophoresis conditions Running temperature: 20 °C Hold: 10 mA, 30 min Hold: 30 mA, 30 min Hold: 60 mA, 105 min 5. Detection ------------ 5.1 Direct detection 5.1.1 Name of direct detection_process Fluorescent staining 5.1.2 Direct detection agents GE Healtcare cy dye DIGE Fluor minimal labelling kit ref: 25-8010-65 Cy3, Cy5, Cy2 : 400 pmol cydye per sample 5.1.3 Additional reagents and buffers DMF 100% Lysine 10mmol 5.1.4 Equipment No specialised equipment. 5.1.5 Direct detection protocol Temperature: 4 °C. Duration: 1 h. Protocol: Each Cy Dye is solved in 5 µl of 100% DMF as a stock solution. 1µl of the stock solution is diluted in 1.5µl of 100% DMF as the labelling solution. 1 µl of the Cy3 or the Cy5 Dye solution is added separatly to 50 µg protein of each "test" and each "control" sample. 2 µl of the Cy2 solution is added to the internal standard protein mixture, which contains 25 µg protein from each sample. Minimal labeling is done for 30 min at 4°C in the dark. Then, 1 µl of the lysine solution is added to each sample to stop the labeling reaction during 10 min still at 4°C in the dark. Each sample are combined according to the design experimental (see section 2.1.1), and DTT is added at a final concentration of 10 mmol for further 10 minutes at 4°C in the dark. The combined samples were supplemented with an equal volume of 2x sample buffer (8M urea, 2M thiourea, 4% (w/v) CHAPS, 1% (v/v) IPG Buffer 3-10 (GE Healthcare) 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment fluorescent scanner 6.1.2 Name of equipment Manufacturer: GE-healtcare Model: Ettan Dige Imager Model number: 11-0036-58 6.1.3 Software Manufacturer: GE-healtcare Model: EDI software Model number: 1.0 6.1.4 Calibration Yes (automatic) 6.1.5 Equipment specific parameters Excitation and emission filters combinations are automatically selected by the software: Cy2 excitation filter 480/30 nm and emission filter 530/40 nm Cy3 excitation filter 540/25 nm and emission filter 595/25 nm Cy5 excitation filter 635/30 nm and emission filter 680/25 nm 6.2 Acquisition Protocol 6.2.1 Image acquisition process exposure time settings gel1 cy2 0.7 cy3 0.15 cy5 0.2 gel2 4 1 2 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) fnsinip310g63347 (format: JPEG) 7.1.2 Dimensions Width: 2283 px Height: 1771 px 7.1.3 Resolution 40 µm/px 7.1.4 Bit-depth 24-bit (TrueColor) 7.1.5 Image location fnsinip310g63347.jpg Link: /download/356/kqj6l6Vx/ 7.1.6 Standard image orientation Yes