Name: Proteome of A549 cells Description: 2D-gel of A549 cells (untreated), ph 4-9 Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2007-05-01 1.1.2 Responsible person or role Affiliation: UFZ-Helmholtz-Centre for Environmental Research (i) Name: PD Dr. Martin von Bergen (ii) Postal address: Department of Proteomics Permoserstr. 15 04318 Leipzig Germany (iii) Email address: martin.vonbergen@ufz.de 1.1.3 Electrophoresis type Two-Dimensional electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: A549 cell lysate 2. Sample type: Control sample 2.1.2 Loading buffer 1. DeStreak Solution (GE Healthcare) with 0.5 % IPG buffer 3-10 NL 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 manufactured using the following reference protocol: Citation: Proteomics,9, page(s) 4920–4933 (2009). URL: not provided. 3.2.3 Physical dimensions X: 180 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: 4:3 3.2.7 Additional substances in gel No additional substance 3.2.8 Gel lane 1 3.2.9 Sample application Lane 1 * Sample: A549 cell lysate * Volume of sample: 500 µg * Loading buffer: DeStreak Solution (GE Healthcare) with 0.5 % IPG buffer 3-10 NL * 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: 300 V, 6 h Gradient: 300-1000 V, 6 h Gradient: 1000-8000 V, 3 h Hold: 8000 V, 9 h 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name reduction 4.1.2 Inter dimension buffer containing 6M urea, 30% glycerol, 4% SDS, 0.05M Tris/HCl, bromophenol blue 4.1.3 Additional reagents dte (20 mg/ml) 4.1.4 Equipment 4.1.5 Protocol Temperature: 20 °C. Duration: 15 min. 4.1.1 Step name alkylation 4.1.2 Inter dimension buffer containing 6M urea, 30% glycerol, 4% SDS, 0.05M Tris/HCl, bromophenol blue 4.1.3 Additional reagents iodoacetamide (25 mg/ml) 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 reference protocol: Citation: Proteomics,9, page(s) 4920–4933 (2009). URL: not provided. 3.2.3 Physical dimensions X: 160 mm Y: 200 mm Z: 1 mm 3.2.4 Physiochemical property range and distribution non linear apparent molecular mass 15 - 200 kDa 3.2.5 Acrylamide concentration 12 % 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: Bisacrylamide Ratio: 30:1 3.2.7 Additional substances in gel No additional substance 3.2.8 Gel lane 1 3.2.9 Sample application Loading method: the strip was placed on the gel and fixed with agarose . 3.3 Protocol 3.3.1 Buffers 0.3% (w/V) TRIS 1.44 % (w/V) Glycine 0.1% (w/V) SDS 3.3.2 Electrophoresis conditions Running temperature: 12 °C Hold: 12 mA, 30 min Hold: 6 mA, 16 h 5. Detection ------------ 5.1 Direct detection 5.1.1 Name of direct detection_process colloidal coomassie 5.1.2 Direct detection agents Coomassie G250 5.1.3 Additional reagents and buffers No additional reagents or buffer 5.1.4 Equipment No specialised equipment. 5.1.5 Direct detection protocol Detection is described in the following reference protocol: Citation: electrophoresis,9, page(s) 255-62 (1988). URL: not provided. 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment flatbed scanner 6.1.2 Name of equipment Manufacturer: GE Healthcare Model: Image scanner II Model number: 18-1170-84 6.1.3 Software Manufacturer: Umax Model: MagicScan Model number: 4.6 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 -default scanner settings 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) a549 big with IDs.png (format: PNG) 7.1.2 Dimensions Width: 1063 px Height: 1094 px 7.1.3 Resolution 150 dpi 7.1.4 Bit-depth 32-bit (TrueColor) 7.1.5 Image location a549 big with IDs.png Link: /download/312/5EClUBD8/ 7.1.6 Standard image orientation Yes