Name: Phakopsora pachyrhizi germinating urediniospore 2-D gel Description: Partial proteome map by 2D gel for Phakopsora pachyrhizi germinating urediniospores. Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2008-05-02 1.1.2 Responsible person or role Affiliation: USDA-ARS (i) Name: Dr. Douglas G. Luster (ii) Postal address: USDA - ARS - FDWSRU 1301 Ditto Avenue Ft. Detrick, MD 21702 (iii) Email address: doug.luster@ars.usda.gov 1.1.3 Electrophoresis type Two-Dimensional electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: Germinating spores 2. Sample type: Test sample 2.1.2 Loading buffer 1. DeStreak Rehydration Solution (GE Healthcare Life Sciences; Piscataway, NJ) with 1% Ampholytes 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 Life Sciences Model: Immobiline DryStrip pH 3-10NL, 13cm Model number: 17-6001-14 Batch number: unknown 3.2.3 Physical dimensions X: 130 mm Y: 3 mm Z: 0.5 mm 3.2.4 Physiochemical property range and distribution non-linear pH 3 - 10 3.2.5 Acrylamide concentration 4% % 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: Bisacrylamide Ratio: 37:1 3.2.7 Additional substances in gel No additional substance 3.2.8 Gel lane 1 3.2.9 Sample application Lane 1 * Sample: Germinating spores * Volume of sample: 400 µg * Loading buffer: DeStreak Rehydration Solution (GE Healthcare Life Sciences; Piscataway, NJ) with 1% Ampholytes * Volume of loading buffer: 250 µL Loading method: rehydration loading. Additional comment: Rehydration at 20degC, 30V for 12h 3.3 Protocol 3.3.1 Buffers MES Running Buffer (Invitrogen; Carlsbad, CA) 3.3.2 Electrophoresis conditions Running temperature: 20 °C Hold: 500 V, 1 h Hold: 1000 V, 1 h Hold: 5000 V, 4 h 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name reduction 4.1.2 Inter dimension buffer 50mM Tris pH 7.0, 6M Urea, 30% (w/v) glycerol, 2% SDS, 10mg/mL DTT 4.1.3 Additional reagents No additional reagent. 4.1.4 Equipment 4.1.5 Protocol Temperature: 25 °C. Duration: 10 min. Protocol: Remove mineral oil from the IPG strips by placing them, gel side up, on a piece of dry filter paper and blotting with a second piece of dry filter paper. Place strip into test tube and add 10mL reduction buffer, rocking gently for 10 minutes on rocker. 4.1.1 Step name alkylation 4.1.2 Inter dimension buffer 50mM Tris pH 7.0, 6M Urea, 30% (w/v) glycerol, 2% SDS, 25mg/mL iodoacetamide 4.1.3 Additional reagents No additional reagent. 4.1.4 Equipment 4.1.5 Protocol Temperature: 25 °C. Duration: 10 min. Protocol: Remove strip from reduction buffer and place in new test tube with 10mL alkylation buffer. Rock gently for 10 minutes on rocker. 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 Laboratories Model: Criterion XL Precast Gel Model number: 345-0127 Batch number: unknown 3.2.3 Physical dimensions X: 140 mm Y: 100 mm Z: 1 mm 3.2.4 Physiochemical property range and distribution linear apparent molecular mass 10 - 220 kDa 3.2.5 Acrylamide concentration 4-12% % (linear) 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: Bisacrylamide Ratio: 37:1 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 placement. Additional comment: Place IPG strip horizontally in IPG well; use BenchMark protein ladder (Invitrogen; Carlsbad, CA). 3.3 Protocol 3.3.1 Buffers MES Running Buffer (Invitrogen; Carlsbad, CA) 3.3.2 Electrophoresis conditions Running temperature: 25 °C Hold: 120 mA, 60 min 5. Detection ------------ 5.1 Direct detection 5.1.1 Name of direct detection_process Coomassie blue staining 5.1.2 Direct detection agents Simply Blue Safestain (Invitrogen; Carlsbad, CA) 5.1.3 Additional reagents and buffers Destaining solution - 4% NaCl 5.1.4 Equipment No specialised equipment. 5.1.5 Direct detection protocol Temperature: 25 °C. Duration: 3 h. Protocol: Rinse gel 3x with 100mL deionized water. Place gel in stain for 3h. Rinse 3x. Destain in salt solution for 16h. 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment camera 6.1.2 Name of equipment Manufacturer: Alpha Innotech Model: FluorChem Model number: 8900 6.1.3 Software Manufacturer: Alpha Innotech Model: FluorChem Model number: 8900 6.1.4 Calibration Yes (automatic) 6.1.5 Equipment specific parameters Visual inspection 6.2 Acquisition Protocol 6.2.1 Image acquisition process Exposure time - 50ms Wavelength - visible light 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) 050208gel (format: TIFF) 7.1.2 Dimensions Width: 1350 px Height: 1102 px 7.1.3 Resolution 150 ppi 7.1.4 Bit-depth 1-bit (monochrome) 7.1.5 Image location GelNoSpots091009.tif Link: /download/121/d2eEGFQc/ 7.1.6 Standard image orientation Yes