Name: pH 4-7 map of bovine placenta protein Description: We analyzed the protein expression of three normal placentae obtained from the afterbirth of AI-derived Korean Native Cattle fetuses born 280 days after insemination. Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2009-08-11 1.1.2 Responsible person or role Affiliation: Professor (i) Name: Jin, Dong IL (ii) Postal address: 79 Daehangno, yoseong-gu, Daejeon, 305-764, Chungnam National University, Korea (iii) Email address: dijin@cnu.ac.kr 1.1.3 Electrophoresis type Two-Dimensional electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: Bovine placenta tissue at late pregnancy 2. Sample type: Standard 2.1.2 Loading buffer 1. 1% SDS, 1 mM PMSF, 1X protease inhibitor cocktail [complete; Roche], 100 mM Tris-HCl(pH 7.0) 2. 7 M urea, 2 M thiourea, 4% CHAPS, 0.1 M DTT, 1 mM PMSF, protease inhibitor, 40 mM Tris-HCl(pH7.0) 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: immobiline Dry strip pH4-7, pH6-9 Model number: 17-1233-01, 17-6001-01 Batch number: unknown 3.2.3 Physical dimensions X: Y: Z: 3.2.4 Physiochemical property range and distribution - 3.2.5 Acrylamide concentration % 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: Ratio: : 3.2.7 Additional substances in gel No additional substance 3.2.8 Gel lane 1 3.2.9 Sample application Lane 1 * Sample: Bovine placenta tissue at late pregnancy * Volume of sample: 1 mg * Loading buffer: 7 M urea, 2 M thiourea, 4% CHAPS, 0.1 M DTT, 1 mM PMSF, protease inhibitor, 40 mM Tris-HCl(pH7.0) * Volume of loading buffer: 450 µL Loading method: rehydration loading. 3.3 Protocol 3.3.1 Buffers No buffer. 3.3.2 Electrophoresis conditions Running temperature: 25 °C Hold: 50 V, 1000 min Hold: 100 V, 100 min Hold: 300 V, 100 min Hold: 600 V, 100 min Hold: 1000 V, 100 min Hold: 3000 V, 100 min Hold: 5000 V, 100 min Hold: 8000 V, 1000 min 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name equilibration 4.1.2 Inter dimension buffer After the first dimensional IEF, IPG gel strip were placed in an equilibration solution (6 M urea, 2% SDS, 50% v/v glycerol, 2.5% acrylamide, 1.875 M Tris-HCl, pH 8.8) containing 5 mM TBP for 20min with gentle shaking. 4.1.3 Additional reagents No additional reagent. 4.1.4 Equipment Manufacturer: GE Healthcare Model: immobiline Dry strip pH4-7, pH6-9 Model number: 17-1233-01, 17-6001-01 4.1.5 Protocol Temperature: 25 °C. Duration: 24 h. 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: unknown Model: unknown Model number: unknown Batch number: unknown 3.2.3 Physical dimensions X: Y: Z: 3.2.4 Physiochemical property range and distribution - 3.2.5 Acrylamide concentration % 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: Ratio: : 3.2.7 Additional substances in gel No additional substance 3.2.8 Gel lane 1 3.2.9 Sample application Loading method: N/A. 3.3 Protocol 3.3.1 Buffers *. 5X SDS Running Buffer (20L) Glycine (amresco, 0167) 1440g SDS (Lauryl Sulfate, Sigma, L-3771) 100g Tris base (amresco, 0826) 300g Water up to 20L 3.3.2 Electrophoresis conditions Running temperature: 25 °C Hold: 30 V, 3 h Hold: 70 V, 20 h 5. Detection ------------ 5.1 Direct detection 5.1.1 Name of direct detection_process Coomassie blue staining 5.1.2 Direct detection agents colloidal Coomassie brilliant blue (CBB) G-250 5.1.3 Additional reagents and buffers Fixer (2L) MeOH (TEDIA, MS-1922) 800ml (net 40%) Phosphoric Acid 100ml (net 5%) Water 1100ml Coomassie (1L) Ammonium sulfate 170g (net 17%) Phosphoric Acid 36ml (net 3%) Coomassie G-250 (Fluka, 27815) 1g MeOH (TEDIA, MS-1922) 340ml (net 34%) Water up to 1L 5.1.4 Equipment 5.1.5 Direct detection protocol Temperature: 25 °C. Duration: 2 d. Protocol: Protocol: 1. At the end of the second dimension run, the gels were removed from the glass plates and washed in deionized water for 1 min. 2. Soaked in fixer for 1 hour. 3. Soaked in CBB buffer for overnight. 4. Washed 3 times in deionized water. 5. Destained overnight in deionized water Detection is described in the following reference protocol: Citation: Proteomics,5, page(s) 4264-4273 (2005). URL: not provided. 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment GS-710 calibrated densitometer scanner 6.1.2 Name of equipment Manufacturer: Bio-rad Model: GS-710 calibrated densitometer Model number: unknown 6.1.3 Software Manufacturer: Swiss Institute for Bioinformatics, Geneva, Switzerland Model: Melanie III Model number: unknown 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 The stained gels were scanned at an optical resolution of 63.5 µm/pixel 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) pH4-7 (format: TIFF) 7.1.2 Dimensions Width: 2404 px Height: 2384 px 7.1.3 Resolution 215.254 ppi 7.1.4 Bit-depth 16-bit (HighColor) 7.1.5 Image location Bovine placenta pH4-7(IDS).tif Link: /download/91/EIJhcnqg/ 7.1.6 Standard image orientation Yes