Name: Proteomes of laboratory-grown B.melitensis strains M5 Description: Proteomes of laboratory-grown B.melitensis strains M5 in the pH 4.0 to 7.0. Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2009-08-30 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 20043077 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. Additional comment: Procedures for 2-DE were carried out according to the manufacturer’s instructions. The sample proteins (1 mg) were separated by isoelectric focusing (IEF) on 18 cm, pH 4-7 linear immobilized pH gradient (IPG) strips. After 12 h of rehydration with 30 V at 20℃ 3.3 Protocol 3.3.1 Buffers No buffer. 3.3.2 Electrophoresis conditions Running temperature: 20 °C 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 ------------ 5.1 Direct detection 5.1.1 Name of direct detection_process Coomassie blue staining 5.1.2 Direct detection agents Coomassie blue staining R35O 1 tablet,1600ml 10% Acetic Acid 5.1.3 Additional reagents and buffers No additional reagents or buffer 5.1.4 Equipment No specialised equipment. 5.1.5 Direct detection protocol Temperature: 100 °C. Duration: 10 min. Detection is described in 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 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 400dpi 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) M5 (format: TIFF) 7.1.2 Dimensions Width: 10000 px Height: 10000 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\M5.tif Link: /download/127/X57GNH7k/ 7.1.6 Standard image orientation Yes