Name: GIPRdn_4-7 Description: Differential proteome analysis of kidney glomeruli of GIPRdn tg vs. wt mice Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2010-09-29 1.1.2 Responsible person or role Affiliation: University of Munich (i) Name: Andreas Blutke (ii) Postal address: Institute of Veterinary Pathology Ludwig-Maximilians-Universitaet München Veterinärstr.13 80539 München Germany. (iii) Email address: blutke@patho.vetmed.uni-muenchen.de 1.1.3 Electrophoresis type Difference gel electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: GIPRdn tg 2. Sample type: Test sample 2.1.2 Loading buffer 1. 2 M Thiourea (Sigma, Germany) 7 M Urea (Roth, Germany) 30 mM Tris (Roth, Germany) 4 % (w/v) CHAPS (3-[Cholamidopropyl]dimethylammonio)-1- propanesulfonate) (Sigma, Germany) Adjust to pH 8.5 with 25 % HCl (Merck, Germany) 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 Bio Sciences Model: Immobiline™ DryStrips, pH 4-7, 18 cm Model number: 17-1233-01 Batch number: ??? 3.2.3 Physical dimensions X: 178 mm Y: 3 mm Z: 0.5 mm 3.2.4 Physiochemical property range and distribution linear pH 4 - 7 3.2.5 Acrylamide concentration 7 % 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: GIPRdn tg * Volume of sample: 120 µg * Loading buffer: 2 M Thiourea (Sigma, Germany) 7 M Urea (Roth, Germany) 30 mM Tris (Roth, Germany) 4 % (w/v) CHAPS (3-[Cholamidopropyl]dimethylammonio)-1- propanesulfonate) (Sigma, Germany) Adjust to pH 8.5 with 25 % HCl (Merck, Germany) * Volume of loading buffer: 50 µL Loading method: cup loading. 3.3 Protocol 3.3.1 Buffers No buffer. 3.3.2 Electrophoresis conditions Running temperature: 20 °C Hold: 0.1 mA, 12 h 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name reduction 4.1.2 Inter dimension buffer 6 M Urea (Roth, Germany) 30 % (v/v) Glycerol (Roth, Germany) 2 % (w/v) SDS (Serva, Germany) 50 mM TrisHCl pH 6.8 4.1.3 Additional reagents 1% DTE 4.1.4 Equipment 4.1.5 Protocol Temperature: 20 °C. Duration: 15 min. Protocol: Shake 15 min 4.1.1 Step name alkylation 4.1.2 Inter dimension buffer 6 M Urea (Roth, Germany) 30 % (v/v) Glycerol (Roth, Germany) 2 % (w/v) SDS (Serva, Germany) 50 mM TrisHCl pH 6.8 4.1.3 Additional reagents 2.5 % iodacetamide 200 µl bromphenol blue 4.1.4 Equipment 4.1.5 Protocol Temperature: 20 °C. Duration: 15 min. Protocol: Shake 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: Separation gel (12 % polyacrylamide) (2 gels) 26.8 ml ddH2O 20 ml 1.5 M TrisHCl pH 8.8 (Roth, Germany) 32 ml Acrylamide / bisacrylamide (37.5:1) (Serva, Germany) 800 μl SDS 10 % (Serva, Germany) 400 μl Ammonium persulfate 10 % (Merck, Germany) 40 μl N,N,N’,N’-Tetramethylethylendiamine (TEMED) (Roth, Germany) Stacking gel (4 % polyacrylamide) (2 gels) 6.1 ml ddH2O 2.5 ml 0.5 M TrisHCl pH 6.8 (Roth, Germany) 1.3 ml Acrylamide / bisacrylamide (37.5:1) (Serva, Germany) 100 μl SDS 10 % (Serva, Germany) 50 μl Ammonium persulfate 10 % (Merck, Germany) 10 μl N,N,N’,N’-Tetramethylethylendiamine (TEMED) (Roth, Germany) 3.2.3 Physical dimensions X: 18 cm Y: 16 cm Z: 0.1 cm 3.2.4 Physiochemical property range and distribution linear apparent molecular mass 100 - 10 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: well loading. 3.3 Protocol 3.3.1 Buffers 25 mM Tris (Roth, Germany) 192 mM Glycine (Roth, Germany) 0.1 % (w/v) SDS (Serva, Germany) 3.3.2 Electrophoresis conditions Running temperature: 20 °C Hold: 30 mA, 30 min Hold: 40 mA, 3 h 5. Detection ------------ 5.1 Direct detection 5.1.1 Name of direct detection_process Fluorescent staining 5.1.2 Direct detection agents DIGE labels 5.1.3 Additional reagents and buffers No additional reagents or buffer 5.1.4 Equipment Manufacturer: GE Healthcare Bio Sciences Model: Typhoon Model number: 63-0055-78 5.1.5 Direct detection protocol Temperature: 20 °C. Duration: 30 min. Protocol: Scanning 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment laser scanner 6.1.2 Name of equipment Manufacturer: GE Healthcare Bio Sciences Model: Typhoon Model number: 63-0055-78 6.1.3 Software Manufacturer: GE Healthcare Bio Sciences Model: Typhoon Model number: 63-0055-78 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 According to manufacturer 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) GIPRdn_4-7 (format: TIFF) 7.1.2 Dimensions Width: 1415 px Height: 1400 px 7.1.3 Resolution 300 dpi 7.1.4 Bit-depth 6-bit 7.1.5 Image location GIPRdn_4-7.tif Link: /download/316/XHD559y6/ 7.1.6 Standard image orientation Yes