Name: 2-DE proteome map of the basic human heart proteins Description: This map described the pH 6-11 region of the human heart proteome. Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2010-02-01 1.1.2 Responsible person or role Affiliation: University College Dublin (i) Name: Julie Polden (ii) Postal address: Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfied, Dublin, Ireland (iii) Email address: julie.polden@ucd.ie 1.1.3 Electrophoresis type Two-Dimensional electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: Left ventricular tissue lysate from human heart 2. Sample type: Control sample 2.1.2 Loading buffer 1. Lysis Buffer: 9.5M urea, 2% CHAPS, 0.8% Pharmalyte pH 3-10, 1% DTT, protease inhibitor cocktail (Complete Mini). Rehydration Buffer: 8M urea, 0.5% CHAPS, 0.2% DTT, 0.2% Pharmaltye pH 6-11, 1.2% Destreak Reagent (GE Healthcare). 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 6-9, 18 cm Model number: 17-6001-97 Batch number: 10008430 3.2.3 Physical dimensions X: 180 mm Y: 3 mm Z: 0.5 mm 3.2.4 Physiochemical property range and distribution linear pH 6 - 11 3.2.5 Acrylamide concentration 0 % 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: Bisacrylamide Ratio: 3:1 3.2.7 Additional substances in gel No additional substance 3.2.8 Gel lane 1 3.2.9 Sample application Lane 1 * Sample: Left ventricular tissue lysate from human heart * Volume of sample: 300 µg * Loading buffer: Lysis Buffer: 9.5M urea, 2% CHAPS, 0.8% Pharmalyte pH 3-10, 1% DTT, protease inhibitor cocktail (Complete Mini). Rehydration Buffer: 8M urea, 0.5% CHAPS, 0.2% DTT, 0.2% Pharmaltye pH 6-11, 1.2% Destreak Reagent (GE Healthcare). * Volume of loading buffer: 100 µL Loading method: paper bridge loading. Additional comment: The IPG strip was passively rehydrated overnight in 350 microlitres rehydration buffer containing 1.2% DeStreak Reagent (GE Healthcare). A sample loading cup was used to ensure the paper bridge remained in contact with the IPG strip. 3.3 Protocol 3.3.1 Buffers No buffer. 3.3.2 Electrophoresis conditions Running temperature: 20 °C Hold: 150 V, 1 h Hold: 300 V, 1 h Hold: 600 V, 1 h Hold: 3500 V, 16 h Gradient: 3500-8000 V, 10 min Hold: 8000 V, 3 h Hold: 100 V, 1 h 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name reduction 4.1.2 Inter dimension buffer 6 M urea, 50 mM TrisCl pH 8.8, 30% glycerol, 2% SDS 4.1.3 Additional reagents 1% DTT 4.1.4 Equipment 4.1.5 Protocol Temperature: 22 °C. Duration: 15 min. Protocol: Strips immersed in individual wells of a rehydration tray and kept shaking for 15 minutes. 4.1.1 Step name alkylation 4.1.2 Inter dimension buffer 6 M urea, 50 mM TrisCl pH 8.8, 30% glycerol, 2% SDS 4.1.3 Additional reagents 4.7% Iodoacetamide 4.1.4 Equipment 4.1.5 Protocol Temperature: 22 °C. Duration: 15 min. Protocol: Strips immersed in individual wells of a rehydration tray and kept shaking for 15 minutes. 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: not provided. URL: http://nationaldiagnostics.com/images/ec890_protocol.pdf Link: http://nationaldiagnostics.com/images/ec890_protocol.pdf 3.2.3 Physical dimensions X: 250 mm Y: 200 mm Z: 1 mm 3.2.4 Physiochemical property range and distribution logarithmic apparent molecular mass 100 - 6 kDa 3.2.5 Acrylamide concentration 12% % 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. Additional comment: IPG strip was placed on top of the slab gel and held in place with Agarose Sealing Solution (0.5% agarose in SDS Electrophoresis Buffer). 3.3 Protocol 3.3.1 Buffers 25mM Tris, 192mM Glycine, 0.1% SDS 3.3.2 Electrophoresis conditions Running temperature: 15 °C Hold: 15 mA, 16 h 5. Detection ------------ 5.1 Direct detection 5.1.1 Name of direct detection_process Silver staining 5.1.2 Direct detection agents http://www.gelifesciences.com/aptrix/upp00919.nsf/Content/FDFA19E77539CA76C1257628001CF9E6/$file/71717700AL.pdf 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: 3 h. Detection is described in the following reference protocol: Citation: Electrophoresis,21, page(s) 3666-3672 (2000). URL: not provided. 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment Calibrated densitometer 6.1.2 Name of equipment Manufacturer: Bio-Rad Model: GS-800 Calibrated Densitometer Model number: 170-7980 6.1.3 Software Manufacturer: Bio-Rad Model: PDQuest Model number: 7.3.1 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 Filter = Green, Light = Transmissive 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) HEART PH6-11 BRIDGE (format: PNG) 7.1.2 Dimensions Width: 909 px Height: 860 px 7.1.3 Resolution 72 dpi 7.1.4 Bit-depth 24-bit (TrueColor) 7.1.5 Image location HEART PH6-11 BRIDGE.png Link: /download/339/FHkTUbyS/ 7.1.6 Standard image orientation Yes