Name: AAA patients Description: Protein expression in the Inferior mesenteric vein (IMV) of patients with AAAs Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2009-03-01 1.1.2 Responsible person or role Affiliation: Researcher (i) Name: Ian Nordon (ii) Postal address: St George's Vascular Institute (iii) Email address: inordon@sgul.ac.uk 1.1.3 Electrophoresis type Difference gel electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: AAA patients 2. Sample type: Test sample 2.1.2 Loading buffer 1. Urea (Amersham Biosciences)8M, 48g Pharmalyte (Amersham Biosciences)0.2%, 2.5(ml) CHAPS (VWR International), 0.5%, 0.5g DTT (Sigma), 2%, 0.4g In 100ml double distilled water. Store at -80ºC 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 manufactured using the following reference protocol: Citation: Industry standrard,n/a, page(s) n/a (2003). URL: http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000149.pdf?pid=PP00000149 Link: http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000149.pdf?pid=PP00000149 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 3 - 10 3.2.5 Acrylamide concentration 8-12 % (linear) 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: Bisacrylamide Ratio: 19:1 3.2.7 Additional substances in gel APS 38ml Glycerol 149ml Temed 44ml Tris-SDS 151ml Water 686ml 3.2.8 Gel lane 1 3.2.9 Sample application Lane 1 * Sample: AAA patients * Volume of sample: 50 µL * Loading buffer: Urea (Amersham Biosciences)8M, 48g Pharmalyte (Amersham Biosciences)0.2%, 2.5(ml) CHAPS (VWR International), 0.5%, 0.5g DTT (Sigma), 2%, 0.4g In 100ml double distilled water. Store at -80ºC * Volume of loading buffer: 320 µL Loading method: rehydration loading. 3.3 Protocol 3.3.1 Buffers Tris-Glycine SDS Running Buffer 3.3.2 Electrophoresis conditions Running temperature: 10 °C Hold: 500 V, 10 min Hold: 500 V, 110 min Hold: 1200 V, 45 min Hold: 2000 V, 30 min Hold: 2500 V, 20 min Hold: 3500 V, 20 min Hold: 3500 V, 10 min Hold: 10000 V, 180 min Hold: 10000 V, 60 min Hold: 500 V, 60 min 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name equilibration 4.1.2 Inter dimension buffer Equilibration buffer Urea Amersham Biosciences 8M 48 SDS Invitrogen 2% 20 Glycerol Invitrogen - 300g Tris pH 8.8 Invitrogen 50mM 33(ml) 4.1.3 Additional reagents No additional reagent. 4.1.4 Equipment 4.1.5 Protocol 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: Industry standrard,n/a, page(s) n/a (2003). URL: http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000149.pdf?pid=PP00000149 Link: http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000149.pdf?pid=PP00000149 3.2.3 Physical dimensions X: 260 mm Y: 210 mm Z: 2 mm 3.2.4 Physiochemical property range and distribution linear apparent molecular mass 200 - 10 kDa 3.2.5 Acrylamide concentration 8-12 % (linear) 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: Bisacrylamide Ratio: 19:1 3.2.7 Additional substances in gel APS 38ml Glycerol 149ml Temed 44ml Tris-SDS 151ml Water 686ml 3.2.8 Gel lane 1 3.2.9 Sample application Loading method: well loading. 3.3 Protocol 3.3.1 Buffers Tris-Glycine SDS Running Buffer 3.3.2 Electrophoresis conditions Running temperature: room temperature (no cooling device). Hold: 600 V, 18.5 h 5. Detection ------------ 5.1 Direct detection 5.1.1 Name of direct detection_process Fluorescent staining 5.1.2 Direct detection agents Cyanine dyes (Cy2/3/5) 5.1.3 Additional reagents and buffers No additional reagents or buffer 5.1.4 Equipment Manufacturer: GE Healthcare Model: Typhoon Model number: 9400 5.1.5 Direct detection protocol Detection is described in the following reference protocol: Citation: Other,n/a, page(s) n/a (2003). URL: http://www.currentprotocols.com/protocol/mb1023 Link: http://www.currentprotocols.com/protocol/mb1023 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: Typhoon Model number: 9400 6.1.3 Software Manufacturer: GE Healthcare Model: ImageQuant Model number: 5.2 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 30 minutes/gel 100 micrometer pixel size Cy2 500nM Cy3 545nM Cy5 550nm 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) AAA (format: TIFF) 7.1.2 Dimensions Width: 2800 px Height: 2080 px 7.1.3 Resolution 100 µm/px 7.1.4 Bit-depth 16-bit (HighColor) 7.1.5 Image location E:\AAA1.tif Link: /download/273/tDhrQpSY/ 7.1.6 Standard image orientation Yes