Name: Mytilus edulis sperm Description: 2-DE proteome map and identification of highly expressed protein spots in sperm of Mytilus edulis Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2007-12-01 1.1.2 Responsible person or role Affiliation: University of Vigo (i) Name: Angel P. Diz (ii) Postal address: Department of Biochemistry, Genetics and Immunology Faculty of Biology University of Vigo Campus Universitario de Vigo s/n 36310 Vigo Spain Tel. +34 986813828 Population Genetics and Cytogenetics Group (XB2) http://webs.uvigo.es/genxb2 (iii) Email address: angel.p.diz@uvigo.es 1.1.3 Electrophoresis type Two-Dimensional electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: Sperm from Mytilus edulis 2. Sample type: Test sample 2.1.2 Loading buffer 1. 7M urea, 2M thiourea, 4% CHAPS, 1% DTT and 1% IPG 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 DryStrip pH 3-10 NL, 24 cm Model number: 17-6002-45 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: 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: Sperm from Mytilus edulis * Volume of sample: 300 µg * Loading buffer: 7M urea, 2M thiourea, 4% CHAPS, 1% DTT and 1% IPG * Volume of loading buffer: 450 µL Loading method: rehydration loading. 3.3 Protocol 3.3.1 Buffers 7M urea, 2M thiourea, 4% CHAPS, 1% DTT and 1% IPG 3.3.2 Electrophoresis conditions Running temperature: 20 °C Hold: 500 V, 2 h Gradient: 500-1000 V, 3 h Hold: 8000 V, 11 h 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name reduction 4.1.2 Inter dimension buffer 50 mM Tris-HCl (pH 8.8), containing 2% (w/v) SDS, 1% (w/v) dithiothreitol (DTT), 6 M urea and 30% (w/v) glycerol 4.1.3 Additional reagents No additional reagent. 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 [50 mM Tris-HCl (pH 8.8), containing 2% (w/v) SDS, 4% (w/v) iodoacetamide, 6 M urea and 30% (w/v) glycerol 4.1.3 Additional reagents No additional reagent. 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 receipe: Acrylamide/bis (30% T, 37.5:1 líquid) H20 mQ 1.5 M Tris-HCL, pH 8.8 10% SDS 10% ammonium persulfate (fresh) TEMED 3.2.3 Physical dimensions X: 22 cm Y: 27 cm Z: 0.1 cm 3.2.4 Physiochemical property range and distribution logarithmic apparent molecular mass 200 - 10 kDa 3.2.5 Acrylamide concentration 12.5 % 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 strip load on the top of 2-DE gel. 3.3 Protocol 3.3.1 Buffers 1× SDS electrophoresis buffer 3.3.2 Electrophoresis conditions Running temperature: 20 °C Hold: 100 V, 45 min Hold: 500 V, 6 h 5. Detection ------------ 5.1 Direct detection 5.1.1 Name of direct detection_process Silver staining 5.1.2 Direct detection agents N/A 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 Temperature: 20 °C. Duration: 5 h. Detection is described in the following reference protocol: Citation: Anal. Chem.,68, page(s) 103-112 (1985). URL: http://pubs.acs.org/doi/abs/10.1021/ac950914h Link: http://pubs.acs.org/doi/abs/10.1021/ac950914h 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: ImageScanner (GE Healthcare) Model number: Unknown 6.1.3 Software Manufacturer: GE Healthcare Model: ImageMaster Platinum Model number: 5 6.1.4 Calibration No (manual) 6.1.5 Equipment specific parameters Default (vendor) parameters. 6.2 Acquisition Protocol 6.2.1 Image acquisition process We have followed manufacturer instructions 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) Mytilus edulis sperm (format: TIFF) 7.1.2 Dimensions Width: 1000 px Height: 1000 px 7.1.3 Resolution 300 ppi 7.1.4 Bit-depth 8-bit (256 colors) 7.1.5 Image location Mytilus sperm gel World 2DPAGE large format.tif Link: /download/402/CQBX1qCO/ 7.1.6 Standard image orientation Yes