Name: HCR_LCR_Cardiac Map Description: Large format 2DE separation of rat cardiac proteins Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2009-02-18 1.1.2 Responsible person or role Affiliation: Liverpool John Moores University (i) Name: Jatin Burniston (ii) Postal address: Research Institute for Sport and Exercise Sciences Liverpool John Moores University Tom Reilly Building Byrom Street Liverpool L3 2ET United Kingdom (iii) Email address: j.burniston@ljmu.ac.uk 1.1.3 Electrophoresis type Two-Dimensional electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: Pooled Standard 2. Sample type: Standard 2.1.2 Loading buffer 1. 7 mol urea, 2 mol thiourea, 2 % (w/v) CHAPS, 20 mmol DTT and 0.5% (v/v) ampholytes 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: 24 cm pH 3-11NL IPG strip Model number: 17-6003-77 Batch number: unknown 3.2.3 Physical dimensions X: 24 cm Y: 3 cm Z: 0.5 cm 3.2.4 Physiochemical property range and distribution Non-linear pH 3 - 11 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: Pooled Standard * Volume of sample: 1000 µg * Loading buffer: 7 mol urea, 2 mol thiourea, 2 % (w/v) CHAPS, 20 mmol DTT and 0.5% (v/v) ampholytes * 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: 20 °C Hold: 300 V, 3 h Hold: 600 V, 3 h Hold: 1000 V, 3 h Gradient: 1000-8000 V, 3 h Hold: 8000 V, 4 h 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name reduction 4.1.2 Inter dimension buffer 50 mmol Tris-HCl pH 8.8, containing 6 mol urea, 30 % v/v glycerol, 2 % w/v SDS and a trace of bromophenol blue 4.1.3 Additional reagents 10 mg/ml dithiothreitol 4.1.4 Equipment 4.1.5 Protocol Temperature: 22 °C. Duration: 15 min. Protocol: Strip equilibration 4.1.1 Step name alkylation 4.1.2 Inter dimension buffer 50 mmol Tris-HCl pH 8.8, containing 6 mol urea, 30 % v/v glycerol, 2 % w/v SDS and a trace of bromophenol blue 4.1.3 Additional reagents 25 mg/ml iodoacetamide 4.1.4 Equipment 4.1.5 Protocol Temperature: 22 °C. Duration: 15 min. Protocol: Strip equilibration 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: Prepare gel mix: for 12.5 % Acrylamide (37.5:1 acrylamide: bis-acrylamide) mix (6 large format gels): 30 % Acrylamide: bis-acrylamide 333 ml 4x Buffer (1.5 M Tris pH 8.8) 250 ml 10 % SDS 8.0 ml 10 % TEMED 1.2 ml *Cool in fridge for 1 h* 10 % (0.1 g/ ml) Ammonium persulphate 8.0 ml 3.2.3 Physical dimensions X: 240 mm Y: 260 mm Z: 1 mm 3.2.4 Physiochemical property range and distribution linear apparent molecular mass 10 - 120 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: well loading. 3.3 Protocol 3.3.1 Buffers 10x Running buffer: 75.5 g Tris base 360.5 g Glycine 25 g SDS ddH2O make up to 2.5 l Dilute 500 ml of 10x Running buffer with 4500 ml ddH2O to make 5 l of 1x Running buffer for lower tank. Dilute 400 ml of 10x Running buffer with 1600 ml ddH2O to make 2 l of 2x Running buffer for upper reservoir 3.3.2 Electrophoresis conditions Running temperature: 20 °C Hold: 70 mA, 30 min Hold: 400 mA, 5 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 blue 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: 22 °C. Duration: 1 h. Protocol: CCB Staining Gel washed 3x 5 minutes in distilled water. Incubate in colloidal Coomassie blue (Bio-safe; Bio-Rad Laboratories) at room temperature for 1 h. De-stained in distilled water overnight at room temperature. 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment Translumination scanner 6.1.2 Name of equipment Manufacturer: Espon Model: Epson 1680 Model number: 1680 6.1.3 Software Manufacturer: Epson Model: Epson Model number: 1680 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 exposure 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) HCR LCR cardiac map (format: TIFF) 7.1.2 Dimensions Width: 1368 px Height: 1050 px 7.1.3 Resolution 100 dpi 7.1.4 Bit-depth 16-bit (HighColor) 7.1.5 Image location HCR LCR Cardiac Map.tif Link: /download/290/K52MtqNm/ 7.1.6 Standard image orientation Yes