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)
-
- Sample name: GIPRdn tg
- Sample type: Test sample
2.1.2 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)
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
13.2.9 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
13.2.9 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 scanner6.1.2 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 manufacturer6.2.2 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-bit7.1.5 Image location
GIPRdn_4-7.tif7.1.6 Standard image orientation
Yes