Name: gel448_BY2_ext_chloromethanol_1mg_A2 Description: 2D-gel of 1 mg of soluble proteins from BY2 cells performed on IPG strips (pH 4.0 to 7.0), followed by SDS-PAGE (12% acrylamide)and colloidal Coomassie Blue staining. Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2008-09-16 1.1.2 Responsible person or role Affiliation: Université Catholique de Louvain-la-Neuve (i) Name: Duby Geoffrey (ii) Postal address: Institut des sciences de la vie Unité FYSA Place Croix du Sud, 4-5 BTE 15 1348 Louvain-la-Neuve Belgique (iii) Email address: geoffrey.duby@uclouvain.be 1.1.3 Electrophoresis type Two-Dimensional electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: 1 mg of soluble proteins from Nicotiana tabacum Bright- Yellow-2 BY2 cells 2. Sample type: Test sample 2.1.2 Loading buffer 1. IEF solubilization buffer : 7 M urea 2 M thiourea 4 % (w/v) CHAPS 0.5 % (v/v) IPG (Immobilized pH Gradient) buffer (pH 4-7) 18 mM DTT 0.002 % (w/v) 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: GE Healthcare Immobiline DryStrip pH 4-7, 24 cm 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: 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: 1 mg of soluble proteins from Nicotiana tabacum Bright- Yellow-2 BY2 cells * Volume of sample: 1 mg * Loading buffer: IEF solubilization buffer : 7 M urea 2 M thiourea 4 % (w/v) CHAPS 0.5 % (v/v) IPG (Immobilized pH Gradient) buffer (pH 4-7) 18 mM DTT 0.002 % (w/v) bromophenol blue * Volume of loading buffer: 450 µL Loading method: rehydration loading. Additional comment: 2 hours of passive rehydratation at 20°C and 10 hours of active rehydratation at 20°C under 30 V 3.3 Protocol 3.3.1 Buffers No buffer. 3.3.2 Electrophoresis conditions Running temperature: 20 °C Gradient: 0-500 V, 2 h Gradient: 500-1000 V, 2 h Gradient: 1000-8000 V, 2 h Hold: 8000 V, 11 h 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name equilibration 4.1.2 Inter dimension buffer first SDS-PAGE equilibration buffer : 50 mM Tris-HCl (pH 8.8) 6 M urea 30 % (v/v) glycerol 2 % (w/v) SDS 0.002 % (w/v) bromophenol blue 4.1.3 Additional reagents 60 mM DTT 4.1.4 Equipment 4.1.5 Protocol Temperature: 20 °C. Duration: 30 min. 4.1.1 Step name equilibration 4.1.2 Inter dimension buffer second SDS-PAGE equilibration buffer : 50 mM Tris-HCl (pH 8.8) 6 M urea 30 % (v/v) glycerol 2 % (w/v) SDS 0.002 % (w/v) bromophenol blue 4.1.3 Additional reagents 130 mM iodoacetamide 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 receipe: 12 % T acrylamide (29:1 acrylamide-bisacrylamide ratio) gel 375 mM Tris-HCl (pH 8.8) 0.1 % SDS 0.005 % (w/v) ammonium persulfate 0.05 % (v/v) TEMED 3.2.3 Physical dimensions X: 255 mm Y: 205 mm Z: 1 mm 3.2.4 Physiochemical property range and distribution logarithmic apparent molecular mass 100 - 10 kDa 3.2.5 Acrylamide concentration 12 % 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: Bisacrylamide Ratio: 29:1 3.2.7 Additional substances in gel No additional substance 3.2.8 Gel lane 1 3.2.9 Sample application Loading method: strip application. Additional comment: The strips were sealed to the gel surface with a 60°C solution of 375 mM Tris-HCl (pH 8.8), 0.1 % (w/v) SDS, 0.002 % (w/v) bromophenol blue, and 0.5 % (w/v) low melting point agarose 3.3 Protocol 3.3.1 Buffers 25 mM Tris-HCl (pH 8.3) 192 mM glycine 0.1 % SDS (w/v) 3.3.2 Electrophoresis conditions Running temperature: 20 °C Hold: 15 mA, 16 h 5. Detection ------------ 5.1 Direct detection 5.1.1 Name of direct detection_process colloidal Coomassie Blue staining 5.1.2 Direct detection agents Coomassie Serva Blue G-250 5.1.3 Additional reagents and buffers No additional reagents or buffer 5.1.4 Equipment Manufacturer: BIO-RAD Model: Dodeca Stainer Model number: 165-3400 5.1.5 Direct detection protocol Temperature: 20 °C. Duration: 3 d. Protocol: After migration, gel was fixed for 3 h in 50 % (v/v) ethanol/2 % (v/v) phosphoric acid, washed for 3 x 30 min in Milli-Q water, and incubated for 1 h in 34 % (v/v) methanol/17 % (w/v) ammonium sulfate/3 % (v/v) phosphoric acid. Coomassie Serva Blue G-250 powder was added to a concentration of 700 mg/l and the gels stained for 3 days and destained with Milli-Q water. Detection is described in the following reference protocol: Citation: Electrophoresis,6, page(s) 427-448 (1985). URL: not provided. 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment scanner 6.1.2 Name of equipment Manufacturer: GE Healthcare Model: ImageScanner Model number: 18-1170-84 6.1.3 Software Manufacturer: GE Healthcare Model: LabScan software Model number: 5.0 6.1.4 Calibration No (manual) 6.1.5 Equipment specific parameters Default (vendor) parameters. 6.2 Acquisition Protocol 6.2.1 Image acquisition process Gel images were acquired on an ImageScanner (GE Healthcare Life Sciences) using LabScan 5 software at 300 dpi and were saved as TIFF and mel files 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) gel448_BY2_ext_chloromethanol_1mg_A2.tiff (format: TIFF) 7.1.2 Dimensions Width: 2982 px Height: 2454 px 7.1.3 Resolution 300 dpi 7.1.4 Bit-depth 16-bit (HighColor) 7.1.5 Image location FigonlineBIG.jpg Link: /download/142/9U6SaLW5/ 7.1.6 Standard image orientation Yes