Name: Analysis of Pig oocyte Description: The protein production of mammalian embryos is known relatively insufficient. Unlike the genome, the proteome itself is forceful reflecting an environmental signal. Until now the lack of sensitivity has remained an uncertain block for the global introduction of proteomics into the field of mammalian embryology. Therefore, we analyzed protein expression level of pig oocyte. Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2010-10-01 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: Pig oocyte 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: pre-cast immobilized dry strips pH3-10 non-linear Model number: 17-1235-01 Batch number: 10035230 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 3-10 % (non-linear) 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 Lane 1 * Sample: Pig oocyte * Volume of sample: 1 mg * Loading buffer: 1% SDS, 1 mM PMSF, 1X protease inhibitor cocktail [complete; Roche], 100 mM Tris-HCl(pH 7.0) * Volume of loading buffer: 100 µL Loading method: rehydration loading. 3.3 Protocol 3.3.1 Buffers anode rehydration buffer (6 M urea, 2 M thiourea, 4% CHAPS, 0.4% DTT, 2% v/v IPG buffer pH 4-7) cathode modified buffer (7 M urea, 2 M thiourea, 4% CHAPS, 2.5% DTT, 10% v/v isopropanol, 5% v/v glycerol, 2% v/v IPG buffer pH 6-11) 3.3.2 Electrophoresis conditions Running temperature: 20 °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, 400 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: Multiphor II IEF system Model number: GE Healthcare Bio-Sciences, Uppsala, Sweden Manufacturer: unknown Model: unknown Model number: unknown 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,5, page(s) 4264-4273 (2005). URL: not provided. 3.2.3 Physical dimensions X: 200 mm Y: 250 mm Z: 1 mm 3.2.4 Physiochemical property range and distribution non-linear apparent molecular mass 10 - 200 kDa 3.2.5 Acrylamide concentration 8-16 % (non-linear) 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: well loading. 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: 20 °C Hold: 30 mA, 1 h Hold: 70 mA, 24 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 Manufacturer: unknown Model: unknown Model number: unknown 5.1.5 Direct detection protocol Temperature: 20 °C. Duration: 1 h. 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: GE Healthcare Model: imagmaster Model number: 5.0 6.1.4 Calibration Yes (automatic) 6.1.5 Equipment specific parameters Default (vendor) parameters. Configuration file: oocyte map Link: /download/91/5AYooBfX/ 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) oocyte map (format: TIFF) 7.1.2 Dimensions Width: 85 px Height: 85 px 7.1.3 Resolution 300 dpi 7.1.4 Bit-depth 8-bit (256 colors) 7.1.5 Image location Pig oocyte (1).tif Link: /download/91/xtLP5tpP/ 7.1.6 Standard image orientation Yes