Name: Pseudomonas putida UW4 proteome reference map Description: Construction of the proteome reference map of a plant growth-promoting bacterium Pseudomonas putida UW4 using 2-D gel and mass spectrometry. Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2007-02-27 1.1.2 Responsible person or role Affiliation: experimenter (i) Name: Zhenyu Cheng (ii) Postal address: 200 University Avenue West Waterloo, Ontario N2L 3V2 Canada (iii) Email address: czhenyu@uwaterloo.ca 1.1.3 Electrophoresis type Two-Dimensional electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: Pseudomonas putida UW4 isolated from rhisosphere 2. Sample type: Standard 2.1.2 Loading buffer 1. 2 M thiourea, 7 M urea, 4% w/v CHAPS, 30 mM Tris pH 8.5, 60 mM DTT, 0.5% v/v IPG pH 4-7 buffer, and a trace of bromophenol blue 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 Model: Immobiline DryStrip Model number: 17-6002-46 Batch number: unknown 3.2.3 Physical dimensions X: 235 mm Y: 3 mm Z: 0.5 mm 3.2.4 Physiochemical property range and distribution linear pH 4 - 7 3.2.5 Acrylamide concentration 4 % 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: Bisacrylamide Ratio: 1:3 3.2.7 Additional substances in gel No additional substance 3.2.8 Gel lane 1 3.2.9 Sample application Lane 1 * Sample: Pseudomonas putida UW4 isolated from rhisosphere * Volume of sample: 1 mg * Loading buffer: 2 M thiourea, 7 M urea, 4% w/v CHAPS, 30 mM Tris pH 8.5, 60 mM DTT, 0.5% v/v IPG pH 4-7 buffer, and a trace of bromophenol blue * Volume of loading buffer: 450 µL Loading method: rehydration loading. 3.3 Protocol 3.3.1 Buffers 2 M thiourea, 7 M urea, 4% w/v CHAPS, 30 mM Tris pH 8.5, 60 mM DTT, 0.5% v/v IPG pH 4-7 buffer, and a trace of bromophenol blue 3.3.2 Electrophoresis conditions Running temperature: 20 °C Hold: 500 V, 1 h Hold: 500.1000 V, 3 h Hold: 1000.3000 V, 3 h Hold: 3000 V, 2 h Hold: 3000.8000 V, 3 h Hold: 8000 V, 10.5 h 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name equilibration 4.1.2 Inter dimension buffer 6M Urea, 2% w/v SDS, 50mM Tris-HCl pH 8.8, 30% v/v Glycerol, 0.002% (w/v) bromophenol blue) 4.1.3 Additional reagents DTT (10mg/ml) 4.1.4 Equipment Manufacturer: VWR Model: rocking platform Model number: 100 4.1.5 Protocol Temperature: 20 °C. Duration: 20 min. Protocol: shaking for 20 minutes 4.1.1 Step name equilibration 4.1.2 Inter dimension buffer 6M Urea, 2% w/v SDS, 50mM Tris-HCl pH 8.8, 30% v/v Glycerol, 0.002% (w/v) bromophenol blue) 4.1.3 Additional reagents 25mg/ml iodoacetamide 4.1.4 Equipment Manufacturer: VWR Model: rocking platform Model number: 100 4.1.5 Protocol Temperature: 20 °C. Duration: 20 min. Protocol: shaking for 20 minutes 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: H2O 33 mL, 30%acrylamide mix 40ml, 1.5M Tris (pH8.8) 25ml, 10% SDS 1ml, 10% ammonium persulfate 1ml, and 0.04ml of TEMED 3.2.3 Physical dimensions X: 24 cm Y: 18 cm Z: 0.1 cm 3.2.4 Physiochemical property range and distribution logarithmic apparent molecular mass 150 - 5 kDa 3.2.5 Acrylamide concentration 12 % 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: well loading. 3.3 Protocol 3.3.1 Buffers 25 mM Tris-HCl pH8.3, 192 mM glycine, 0.1%SDS 3.3.2 Electrophoresis conditions Running temperature: 10 °C Hold: 17.5 mA, 6 h 5. Detection ------------ 5.1 Direct detection 5.1.1 Name of direct detection_process Coomassie blue staining 5.1.2 Direct detection agents 1L of 2 g/L coomassie G-250, 1L of 1M sulfuric acid, 220 mL of 10M sodium hydroxide, 310 mL of 100% TCA 5.1.3 Additional reagents and buffers No additional reagents or buffer 5.1.4 Equipment Manufacturer: VWR Model: rocking platform Model number: 100 5.1.5 Direct detection protocol Temperature: 20 °C. Duration: 18 h. Protocol: shaking overnight 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment fluorescent scanner 6.1.2 Name of equipment Manufacturer: GE Healthcare Model: Typhoon Model number: 9400 6.1.3 Software Manufacturer: GE Healthcare Model: Typhoon scanner control Model number: 3.0 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 excitation wavelength of 633nm and without an emission filter PMT 600 scan time 15 min 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) Figure 1 (format: TIFF) 7.1.2 Dimensions Width: 2294 px Height: 1845 px 7.1.3 Resolution 254 dpi 7.1.4 Bit-depth 8-bit (256 colors) 7.1.5 Image location Cheng_database.tif Link: /download/28/m0zJFiAu/ 7.1.6 Standard image orientation Yes