Name: Reference map for Corynebacterium glutamicum ATCC14067, pH 4.5-5.5 Description: Cytoplasmic protein extract from cells of Corynebacterium glutamicum ATCC14067, pH 4.5-5.5 Reference: PROTEOMICS (2007) 7(23): 4317-4322 ( doi:10.1002/pmic.200700269 Link: http://dx.doi.org/10.1002/pmic.200700269) Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2003-04-01 1.1.2 Responsible person or role Affiliation: Hokkaido University (i) Name: Prof. Dr. Atsushi Yokota (ii) Postal address: Laboratory of Microbial Physiology Research Faculty of Agriculture Hokkaido University Sapporo, Hokkaido 060-8589, Japan (iii) Email address: yokota@chem.agr.hokudai.ac.jp 1.1.3 Electrophoresis type Two-Dimensional electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: Cytoplasmic protein extact from cells of Corynebacterium glutamicum ATCC14067 2. Sample type: Test sample 3. URL: http://dx.doi.org/10.1002/pmic.200700269 Link: http://dx.doi.org/10.1002/pmic.200700269 2.1.2 Loading buffer 1. 8 M urea, 4% w/v CHAPS, 65 mM DTT, 40 mM Tris-HCl (pH 8.0), 2% v/v IPG 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 Bio-Sciences Corp. Model: Immobiline DryStrip 4.5-5.5 Model number: 17-6001-85 Batch number: unknown 3.2.3 Physical dimensions X: 180 mm Y: 3 mm Z: 0.5 mm 3.2.4 Physiochemical property range and distribution linear pH 4.5 - 5.5 3.2.5 Acrylamide concentration 4 % 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: Bisacrylamide Ratio: 32:1 3.2.7 Additional substances in gel No additional substance 3.2.8 Gel lane 1 3.2.9 Sample application Lane 1 * Sample: Cytoplasmic protein extact from cells of Corynebacterium glutamicum ATCC14067 * Volume of sample: 100 µg * Loading buffer: 8 M urea, 4% w/v CHAPS, 65 mM DTT, 40 mM Tris-HCl (pH 8.0), 2% v/v IPG buffer, and a trace of bromophenol blue * Volume of loading buffer: 400 µL Loading method: rehydration loading. 3.3 Protocol 3.3.1 Buffers No buffer. 3.3.2 Electrophoresis conditions Running temperature: 20 °C Hold: 60 V, 0.1 h Hold: 150 V, 1 h Hold: 500 V, 2 h Gradient: 500-3500 V, 18 h 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name equilibration 4.1.2 Inter dimension buffer Buffer A: 6 M urea, 30% v/v glycerol, 1% w/v SDS, 16.25 mM DTT, 50 mM Tris-HCl, pH 6.8. Buffer B: buffer A in which DTT was replaced with 45 mg/mL iodoacetamide. 4.1.3 Additional reagents No additional reagent. 4.1.4 Equipment Manufacturer: GE Healthcare Bio-Sciences Corp. Model: Multiphor II Model number: 18-1018-06 4.1.5 Protocol Protocol: 10 min in Buffer A and then 10 min in Buffer B 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: GE Healthcare Bio-Sciences Corp. Model: ExcelGel XL SDS 12-14 Model number: 17-1236-01 Batch number: unknown 3.2.3 Physical dimensions X: 245 mm Y: 180 mm Z: 0.5 mm 3.2.4 Physiochemical property range and distribution logarithmic apparent molecular mass 12 - 116 kDa 3.2.5 Acrylamide concentration 12-14 % (unknown) 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_transfer. 3.3 Protocol 3.3.1 Buffers 0.12 M Tris Acetate (pH 6.6), 1g/L SDS 3.3.2 Electrophoresis conditions Running temperature: room temperature (no cooling device). Hold: 1000 V, 45 min Hold: 1000 V, 4 min Hold: 1000 V, 160 min 5. Detection ------------ 5.1 Direct detection 5.1.1 Name of direct detection_process Fluorescent staining 5.1.2 Direct detection agents SYPRO Ruby Protein Gel Stain (Bio-Rad Laboratories, Inc.) 5.1.3 Additional reagents and buffers fixing solution: 10% (v/v) Ethanol, 7% (v/v) Acetic acid 5.1.4 Equipment No specialised equipment. 5.1.5 Direct detection protocol Protocol: according to the instruction from the manufacturer: http://wolfson.huji.ac.il/purification/PDF/Stains/BioRadSyproRubinProtStains.pdf Detection is described in the following reference protocol: Citation: not provided. URL: http://www.proteomic.org/html/staining.html Link: http://www.proteomic.org/html/staining.html 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment fluorescent scanner 6.1.2 Name of equipment Manufacturer: GE Healthcare Bio-Sciences Corp. Model: FluorImager 595 Model number: 63001545 6.1.3 Software Manufacturer: GE Healthcare Bio-Sciences Corp. Model: ImageQuant Model number: unknown 6.1.4 Calibration Yes (automatic) 6.1.5 Equipment specific parameters unknown 6.2 Acquisition Protocol 6.2.1 Image acquisition process unknown 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) CORGL-ATCC14067-4.5-5.5 (format: JPEG) 7.1.2 Dimensions Width: 2008 px Height: 1606 px 7.1.3 Resolution 300 dpi 7.1.4 Bit-depth 16-bit (HighColor) 7.1.5 Image location http://world-2dpage.expasy.org/0001/html/data/gifs/yokota_0001/CORGL-ATCC14067-4.5-5.5.jpg Link: http://world-2dpage.expasy.org/0001/html/data/gifs/yokota_0001/CORGL-ATCC14067-4.5-5.5.jpg 7.1.6 Standard image orientation Yes