Name: Proteome reference map for Xanthomonas oryzae pv. oryzae ZJ173 (pH 3-10) Description: Construction of the proteome reference map(pH 3-10) for the plant-pathogenic bacterium Xanthomonas oryzae pv. oryzae ZJ173 using 2-D gel and MALDI-TOF-TOF MS. Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2012-04-13 1.1.2 Responsible person or role Affiliation: experimenter (i) Name: Shu Xu (ii) Postal address: College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China (iii) Email address: 2009202039@njau.edu.cn 1.1.3 Electrophoresis type Two-Dimensional electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: Xanthomonas oryzae pv. oryzae 2. Sample type: Standard 2.1.2 Loading buffer 1. 8 M urea, 2 M thiourea, 4% CHAPS (w/v), 0.2% (w/v) Bio-Lyte (pH 3-10), 0.001% (w/v) bromophenol blue, 1 mM PMSF, and 2 mM EDTA 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: BioRad Model: IPG strip 24 cm; pH 3-10 Model number: 163-2043 Batch number: unknown 3.2.3 Physical dimensions X: 24.7 cm Y: 0.33 cm Z: 0.05 cm 3.2.4 Physiochemical property range and distribution nonlinear pH 3 - 10 3.2.5 Acrylamide concentration 4 % 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: Bisacrylamide Ratio: 32.3:1 3.2.7 Additional substances in gel No additional substance 3.2.8 Gel lane 1 3.2.9 Sample application Lane 1 * Sample: Xanthomonas oryzae pv. oryzae * Volume of sample: 1500 µg * Loading buffer: 8 M urea, 2 M thiourea, 4% CHAPS (w/v), 0.2% (w/v) Bio-Lyte (pH 3-10), 0.001% (w/v) bromophenol blue, 1 mM PMSF, and 2 mM EDTA * Volume of loading buffer: 480 µL Loading method: rehydration loading. 3.3 Protocol 3.3.1 Buffers No buffer. 3.3.2 Electrophoresis conditions Running temperature: 20 °C Gradient: 0-100 V, 0.5 h Gradient: 100-250 V, 0.5 h Gradient: 250-500 V, 0.5 h Hold: 1000 V, 1 h Gradient: 1000-10000 V, 5 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, 2% SDS, 20% (v/v) glycerol, 0.375 M Tris-HCl pH 8.8, and 20mg/ml DTT 4.1.3 Additional reagents No additional reagent. 4.1.4 Equipment Manufacturer: Nanjing University Model: rocking platform Model number: unknown 4.1.5 Protocol Temperature: 20 °C. Duration: 15 min. Protocol: shaking for 15 min 4.1.1 Step name alkylation 4.1.2 Inter dimension buffer 6M urea, 2% SDS, 20% (v/v) glycerol, 0.375 M Tris-HCl pH 8.8, and 25mg/ml iodoacetamide 4.1.3 Additional reagents No additional reagent. 4.1.4 Equipment Manufacturer: Nanjing University Model: rocking platform Model number: unknown 4.1.5 Protocol Temperature: 20 °C. Duration: 15 min. Protocol: shaking for 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 receipe: For each independent experiment, six gels were manufactured using the following receipe: 161.2 ml Milli-Q water, 124.8 ml 30 % acrylamide/Bis solution, 98.8 ml buffer (1.5 M Tris-HCl pH 8.8, 4% SDS), 5.8 ml 10% APS solution, 260 µl TEMED 3.2.3 Physical dimensions X: 24 cm Y: 18 cm Z: 0.1 cm 3.2.4 Physiochemical property range and distribution linear apparent molecular mass 150 - 20 kDa 3.2.5 Acrylamide concentration 10 % 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: Bisacrylamide Ratio: 37.5:1 3.2.7 Additional substances in gel No additional substance 3.2.8 Gel lane 1 3.2.9 Sample application Loading method: IPG strip placing. 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: 10 °C Hold: 60 mA, 0.25 h Hold: 120 mA, 5 h 5. Detection ------------ 5.1 Direct detection 5.1.1 Name of direct detection_process Coomassie blue staining 5.1.2 Direct detection agents Fixation solution(2L): 40% methanol, 10% acetic acid Staining solution(2L): Coomassie G-250 2.4 g, phosphoric acid 232ml, ammonium sulfate 200g , methanol 400 ml 5.1.3 Additional reagents and buffers No additional reagents or buffer 5.1.4 Equipment Manufacturer: BioRad Model: GS-800 scanner Model number: 170-7981 5.1.5 Direct detection protocol Detection is described in the following reference protocol: Citation: Electrophoresis,25, page(s) 1327-1333 (2004). URL: not provided. 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment Calibrated Densitometer 6.1.2 Name of equipment Manufacturer: BioRad Model: GS-800 scanner Model number: 170-7981 6.1.3 Software Manufacturer: BioRad Model: Quantity One Model number: 4.62 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 'gel' and 'Coomassie Blue' were chosen, and image acquisition was conducted after preview scan. 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) 3-10 (format: TIFF) 7.1.2 Dimensions Width: 2992 px Height: 2289 px 7.1.3 Resolution 400 dpi 7.1.4 Bit-depth 8-bit (256 colors) 7.1.5 Image location C:\\\\Users\\\\Administrator\\\\Desktop\\\\3-10.tif Link: /download/456/uiQWx25O/ 7.1.6 Standard image orientation Yes