Name: Fusarium graminearum strain 453 reference map Description: Proteome reference map of the nivalenol producer strain Fusarium graminearum 453 using a 2D-DiGE protocol. In order to obtain a more complete map, the Fusarium graminearum 453 strain was grown in two different culture media, one containing Glutamic acid as source of Nitrogen and the other containing Agmatin as Nitrogen source. Both media are considered to be good inducer of toxin production. Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2010-09-18 1.1.2 Responsible person or role Affiliation: Centre de Recherce Public Gabriel Lippmann (i) Name: Dr. Tommaso Serchi (ii) Postal address: 41, rue du Brill, L4422 Belvaux, Luxembourg (iii) Email address: serchi@lippmann.lu 1.1.3 Electrophoresis type Difference gel electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: Fusarium graminearum (strain453) mycelium, isolated in Luxembourg 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, DeStreak reagent (GE Healthecare) 0.005% v/v, 0.2% v/v IPG pH 3-10 (BioRad) 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: BioRad Model: ReadyStrip IPG Strips, pH 3–10 nonlinear, 24 cm, Model number: 163-2043 Batch number: unknown 3.2.3 Physical dimensions X: 240 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: 32.5:1 3.2.7 Additional substances in gel No additional substance 3.2.8 Gel lane 1 3.2.9 Sample application Lane 1 * Sample: Fusarium graminearum (strain453) mycelium, isolated in Luxembourg * Volume of sample: 90 µg * Loading buffer: 2 M thiourea, 7 M urea, 4% w/v CHAPS, 30 mM Tris pH 8.5, DeStreak reagent (GE Healthecare) 0.005% v/v, 0.2% v/v IPG pH 3-10 (BioRad) buffer, and a trace of bromophenol blue * Volume of loading buffer: 500 µL Loading method: rehydration loading. 3.3 Protocol 3.3.1 Buffers No buffer. 3.3.2 Electrophoresis conditions Running temperature: 25 °C Hold: 30 V, 3 h Gradient: 30-200 V, 2 h Hold: 200 V, 3 h Gradient: 200-1000 V, 4 h Hold: 1000 V, 2 h Gradient: 1000-5000 V, 4 h Hold: 5000 V, 1 h Gradient: 5000-10000 V, 4 h Hold: 10000 V, 6 h 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name reduction 4.1.2 Inter dimension buffer The buffer used in the reduction is provided by Serva Electrophoresis. The buffer needs to be added 7.2 g of Urea and 200 mg of DTT per 20 mL of equilibration solutuion. The solution provided is already supplemented with bromophenol blue. 4.1.3 Additional reagents Urea (approximately 6 M) DTT (approximately 54 mM) 4.1.4 Equipment 4.1.5 Protocol Temperature: 25 °C. Duration: 15 min. Protocol: Put the strip in a tray face up and add 10 mL of equilibration solution (10 mL per strip). Shacke gently. 4.1.1 Step name alkylation 4.1.2 Inter dimension buffer The buffer used in the reduction is provided by Serva Electrophoresis. The buffer needs to be added 7.2 g of Urea and 500 mg of IAA per 20 mL of euilibration solutuion. The solution provided is already supplemented with bromophenol blue. 4.1.3 Additional reagents Urea (approximately 6 M) Iodio-Acetamide (approximately 140 mM) 4.1.4 Equipment 4.1.5 Protocol Temperature: 25 °C. Duration: 15 min. Protocol: Put the strip in a tray face up and add 10 mL of equilibration solution (10 mL per strip). Shacke gently. 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: Serva Electrophoresis Model: 2D Gel DALTtwelve 12.5 % Kit Model number: 43319.01 Batch number: unkown 3.2.3 Physical dimensions X: 260 mm Y: 200 mm Z: 1 mm 3.2.4 Physiochemical property range and distribution linear apparent molecular mass 5 - 200 kDa 3.2.5 Acrylamide concentration 12.5 % 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: Bisacrylamide Ratio: 32.5:1 3.2.7 Additional substances in gel No additional substance 3.2.8 Gel lane 1 3.2.9 Sample application Loading method: Loading method: IPG transfer.. 3.3 Protocol 3.3.1 Buffers The buffers are provided by the Serva Electrophoresis company. They consist of two bags containing one the dry powder for the cathode and one the dry powder for the anode. Dilution was performed following the manufacturer's instruction: briefly, the cathode buffer is to be diluted in 2.5 L of milliQ water; the anode buffer is to be diluted in 7.5 L of milliQ water. 3.3.2 Electrophoresis conditions Running temperature: 25 °C Hold: 10 mA, 2 h Hold: 30 mA, 14 h 5. Detection ------------ 5.1 Direct detection 5.1.1 Name of direct detection_process Fluorescent staining 5.1.2 Direct detection agents Cy2, Cy3 and Cy5 - CyDyes minimal labeling kit (25 nmol)- GE Healthcare 5.1.3 Additional reagents and buffers DMF 100% 10 mM Lysin 5.1.4 Equipment Manufacturer: GE Healthcare Model: Typhoon 9400 Model number: unknown 5.1.5 Direct detection protocol Temperature: 20 °C. Duration: 1 h. Protocol: 1) A volume of sample equivalent to 30 μg of proteins was added to a microfuge tube. 2) 240 pmol of the appropriate dye were added to the microfuge tube containing the protein sample. 3) Dye and protein sample were mixed thoroughly by pipetting and vortexing. 4) The tube was then centrifuged briefly in a microcentrifuge to collect the solution at the bottom of the tube. 5) The labeling reaction was carried out leaving the tube on ice for 30 min in the dark. 6) The reaction was stopped adding 1 μl of 10 mM lysine to the reaction tube. 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment laser scanner 6.1.2 Name of equipment Manufacturer: GE Healthcare Model: Typhoon 9400 Model number: unknown 6.1.3 Software Manufacturer: GE Healthcare Model: Typhoon Scanner Control Model number: unknown 6.1.4 Calibration Yes (automatic) 6.1.5 Equipment specific parameters Cy2 excitation filter 480/30nm and emission filter 530/40nm Cy3 excitation filter 540/25nm and emission filter 595/25nm Cy5 excitation filter 635/30nm and emission filter 680/25nm 6.2 Acquisition Protocol 6.2.1 Image acquisition process 1) Turn on the Typhoon and leave the instrument to warm up for at least 30 min prior to scanning. 2) Ensure that the gel glass plates are clean, dry and free from lint. 3)Select scan area 4)Select the appropriate wavelenths and adjust the intensity of the lasers in a way to have the maximum signal with no saturation. The adjustemnt can be done using a scanning resolution of 1mm 5)After calibration scan the gel setting the resolution to 100 um. 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) Cy3 (format: TIFF) 7.1.2 Dimensions Width: 741 px Height: 1000 px 7.1.3 Resolution 254 ppi 7.1.4 Bit-depth 8-bit (256 colors) 7.1.5 Image location Cy3.tiff Link: /download/514/F6dUF4Ox/ 7.1.6 Standard image orientation Yes 7. Image -------- 7.1.1 Image name (or id) Cy5 (format: TIFF) 7.1.2 Dimensions Width: 741 px Height: 1000 px 7.1.3 Resolution 254 ppi 7.1.4 Bit-depth 8-bit (256 colors) 7.1.5 Image location Cy5.tiff Link: /download/514/LUu4Z1f3/ 7.1.6 Standard image orientation Yes 7. Image -------- 7.1.1 Image name (or id) Cy2 (format: TIFF) 7.1.2 Dimensions Width: 741 px Height: 1000 px 7.1.3 Resolution 254 ppi 7.1.4 Bit-depth 8-bit (256 colors) 7.1.5 Image location CY2.tiff Link: /download/514/gejgoMF4/ 7.1.6 Standard image orientation Yes