Name: 2-DE and 2-DE Western blotting of soluble proteins of B. melitensis M5 Description: 2-DE and 2-DE Western blotting of soluble proteins of B. melitensis M5 Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2009-08-31 1.1.2 Responsible person or role Affiliation: Department of Immunology (i) Name: Beijing Institute of Microbiology & Epidemiology (ii) Postal address: No.20 Dongda street, Fengtai District, Beijing 100071 (iii) Email address: xiaozzp@eyou.com 1.1.3 Electrophoresis type Two-Dimensional electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: Soluble proteins of M5 2. Sample type: Test sample 2.1.2 Loading buffer 1. 8M urea, 1% DTT, 4% CHAPS, suitable protease inhibitor 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: Amersham pharmaci Model: IPG 4.0-7.0 Model number: 17-1233-01 Batch number: Amersham pharmaci 20043081 3.2.3 Physical dimensions X: Y: Z: 3.2.4 Physiochemical property range and distribution - 3.2.5 Acrylamide concentration % 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: Ratio: : 3.2.7 Additional substances in gel No additional substance 3.2.8 Gel lane 1 3.2.9 Sample application Lane 1 * Sample: Soluble proteins of M5 * Volume of sample: 1 mg * Loading buffer: 8M urea, 1% DTT, 4% CHAPS, suitable protease inhibitor * Volume of loading buffer: 350 µL Loading method: rehydration loading. 3.3 Protocol 3.3.1 Buffers No buffer. 3.3.2 Electrophoresis conditions Running temperature: 20 °C Hold: 30 V, 10.00 h Hold: 500 V, 30 min Hold: 2000 V, 30 min Hold: 5000 V, 30 min Gradient: 5000-10000 V, 2 h Hold: 10000 V, 8 h 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name reduction 4.1.2 Inter dimension buffer 6 M urea,30% glycerol,1%DTT 4.1.3 Additional reagents No additional reagent. 4.1.4 Equipment 4.1.5 Protocol Temperature: 20 °C. Duration: 15 min. 4.1.1 Step name alkylation 4.1.2 Inter dimension buffer 0.05 M Tris -HCl , pH 8.8 ，6 M urea, 30% (w/v) glycerol and 2% (w/v) SDS,4%iodoacetamide. 4.1.3 Additional reagents No additional reagent. 4.1.4 Equipment 4.1.5 Protocol Temperature: 20 °C. Duration: 15 min. 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 reference protocol: Citation: Chinese J ourna1 of Zoonoses,23(12), page(s) 1172-1175 (2007). URL: http://www.cqvip.com Link: http://www.cqvip.com 3.2.3 Physical dimensions X: Y: Z: 3.2.4 Physiochemical property range and distribution - 3.2.5 Acrylamide concentration % 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: Ratio: : 3.2.7 Additional substances in gel No additional substance 3.2.8 Gel lane 1 3.2.9 Sample application Loading method: paper bridge loading. 3.3 Protocol 3.3.1 Buffers 25 mM Tris-HCl, pH 8.3, 192 mM glycine, 0.1% SDS 3.3.2 Electrophoresis conditions Running temperature: 20 °C Hold: 20 mA, 45 min Hold: 30 mA, 4 h 5. Detection ------------ 5.2 Indirect detection 5.2.1 Name of direct detection_process N/A 5.2.2 Transfer medium N/A Manufacturer: N/A Model: N/A Model number: N/A 5.2.3 Detection medium N/A Manufacturer: N/A Model: N/A Model number: N/A 5.2.4 Indirect detection agents N/A 5.2.5 Additional reagents and buffers No additional reagents or buffer 5.2.6 Equipment No specialised equipment. 5.2.7 Indirect detection protocol 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment laser scanner 6.1.2 Name of equipment Manufacturer: Amersham pharmaci Model: Imagescanner Model number: 18-1134-45 6.1.3 Software Manufacturer: Amersham pharmaci Model: ImageMaster TM 2D platinum Model number: version5 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 Western blotting was performed according to the method of Towbin et al. Proteins were transferred to PVDF membranes using TE77 ECL semi-dry transfer unit (0.8 mA/cm2, 1h). After completion of transfer, non-specific binding sites on the membranes were blocked for 90 min with 5% skimmed milk in TBS at 37℃. Then, PVDF membranes were incubated with primary antibody, rabbit anti-B. melitensis M5 (the agglutination titer of the pooled sera was 1:3200), diluted 1:50 in TBS containing 5% skimmed milk for 1 h at 37℃ on a gentle shaker. The membranes were rinsed in 0.1% Tween20 TBS three times, 10 min each, and incubated with goat anti-rabbit-HRP at a dilution of 1:10000 in TBS containing 5% skimmed milk for 1 h at 37℃. After washing, the blots were developed using ECL Western blotting detection reagents. The specific immunogenic protein pattern was visualized on an X-ray film. 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) sM5 (format: TIFF) 7.1.2 Dimensions Width: 1000 px Height: 1000 px 7.1.3 Resolution 400 dpi 7.1.4 Bit-depth 32-bit (TrueColor) 7.1.5 Image location C:\Documents and Settings\immun323.AMMS-49CC6649A4\桌面\soluble.doc Link: /download/127/LtGklnha/ 7.1.6 Standard image orientation Yes