Name: CCB5_vs_CCB90_repl1
Description: This gel is composed of a cytosolic fraction of a Cy-5 labeled 5min cortisol-BSA stimulation (biological replicate 1), a cytosolic fraction of a Cy-3 labeled 90min cortisol-BSA stimulation (biological replicate 1) and the Cy-2 labeled internal standard.
Version: MIAPE: Gel Electrophoresis 1.4
1. General features
1.1.1 Date Stamp
2010-07-21
1.1.2 Responsible person or role
Affiliation: Institute of Immunology, Centre de Recherche Public de la Santé / National Public Health Laboratory
(i) Name: Claude P. Muller
(ii) Postal address: Institute of Immunology,
Centre de Recherche Public de la Santé / National Public Health Laboratory,
20A rue Auguste Lumière,
L-1950 Luxembourg,
Grand-Duchy of Luxembourg
(iii) Email address: claude.muller@CRP-SANTE.LU
1.1.3 Electrophoresis type
Difference gel electrophoresis
2. Sample
2.1.1 Sample Name(s)
-
- Sample name: CCB90_1(Cy3)+CCB5_1(Cy5)+Internal standard (Cy2)
- Sample type: Test sample
2.1.2 Loading buffer
- Rehydration buffer composition: 2M thiourea, 7M urea, 2% (w/v) CHAPS, 1% IPG buffer pH 3-10 NL, 12uL/mL DeStreak Reagent
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 Immobiline (TM) DryStrips NL pH 3-10
Model number: 17-6002-44
Batch number: unknown
3.2.3 Physical dimensions
X: 240 mm
Y: 3 mm
Z: 0.5 mm
3.2.4 Physiochemical property range and distribution
non-linear pH 3 - 10
3.2.5 Acrylamide concentration
4 %
3.2.6 Acrylamide : Crosslinker ratio
Crosslinker: Bisacrylamide
Ratio: 32:1
3.2.7 Additional substances in gel
No additional substance
3.2.8 Gel lane
13.2.9 Sample application
Lane 1
- Sample: CCB90_1(Cy3)+CCB5_1(Cy5)+Internal standard (Cy2)
- Volume of sample: 150 µg
- Loading buffer: Rehydration buffer composition: 2M thiourea, 7M urea, 2% (w/v) CHAPS, 1% IPG buffer pH 3-10 NL, 12uL/mL DeStreak Reagent
- 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: room temperature (no cooling device).
Hold: 40 V, 2 h
Hold: 70 V, 2 h
Hold: 300 V, 2 h
Hold: 1 kV, 4 h
Hold: 8 kV, 4 h
Hold: 8 kV, 9 h
4. Inter-dimension Process
4.1 Protocol
4.1.1 Step name
reduction
4.1.2 Inter dimension buffer
Equilibration buffer (Gel Company) supplemented with 8M urea and 65mM DTT
4.1.3 Additional reagents
No additional reagent.
4.1.4 Equipment
4.1.5 Protocol
Temperature: 21 °C.
Duration: 15 min.
Protocol:
IPG strips were incubated in reduction buffer for 15min at RT on an orbital shaker set to 30rpm.
4.1.1 Step name
alkylation
4.1.2 Inter dimension buffer
Equilibration buffer (Gel Company) supplemented with 8M urea and 135mM IAA
4.1.3 Additional reagents
No additional reagent.
4.1.4 Equipment
4.1.5 Protocol
Temperature: 21 °C.
Duration: 15 min.
Protocol:
IPG strips were incubated at RT for 15min in reduction buffer on an orbital shaker set to 30rpm.
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 purchased precast.
Manufacturer: Gel Company
Model: 2D DALT NF 12.5% pre cast polyacrylamid gels
Model number: 1019-21
Batch number: unknown
3.2.3 Physical dimensions
X: 260 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
12.5 %
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
13.2.9 Sample application
Loading method: Loading from IPG strip.
Additional comment: IPG strip was inserted on top of the slab gel and hold in place using low melting agarose (Gel Company)
3.3 Protocol
3.3.1 Buffers
Anode Buffer:
anode buffer powder (Gel Company dissolved in 7.5 L of deionized water)
3.3.2 Electrophoresis conditions
Running temperature: 25 °C
Hold: 5 mA, 2 h
Hold: 16 mA, 14 h
5. Detection
5.1 Direct detection
5.1.1 Name of direct detection_process
Fluorescent staining
5.1.2 Direct detection agents
CyDye DIGE Fluor minimal labelling (GE Healtcare)
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: 4 °C.
Duration: 1 h.
Protocol:
Cy-dyes were reconstituted in 100% DMF to a stock solution of 1000pmol/µL. A working solution of 400pmol/µL was prepared by further diluting the stock solution in 100%DMF. The sample pH was adjusted to 8.5 using 100mM NaOH and proteins were labeled with 8pmol of Cy-dye/µg of protein i.e. by adding 1µL Cy-dye working solution to 50µg of protein sample. Samples were incubated for 30min in the dark and on ice. The labeling reaction was quenched by adding 1µL of 10mM L-Lysine per labeling reaction and incubating 30min in the dark and on ice. The protein samples run on the same gel were pooled and a volume equivalent to 50µg of internal standard was added.
6. Image Acquisition
6.1 Acquisition Equipment
6.1.1 Type of equipment
fluorescent scanner6.1.2 Name of equipment
Manufacturer: GE Healthcare
Model: Typhoon 9400 Varaible Mode Imager
Model number: unknown
6.1.3 Software
Manufacturer: GE Healthcare
Model: Tyhoon Scanner Software
Model number: v5.0
6.1.4 Calibration
Yes (automatic)
6.1.5 Equipment specific parameters
Gels containing Cy-labeled proteins were scanned at 100 μm resolution using a PMT voltage of 520 V.Lava purple stained preparative gels were scanned at 100 μm resolution and a PMT voltage of 400 V.
6.2 Acquisition Protocol
6.2.1 Image acquisition process
For gels conatining Cy-labeled proteins the following excitation/emission wavelengths were used:Cy2 488 nm/520 nm BP40;
Cy3 532 nm/580 nm BP 30;
Cy5 633 nm/670 nm BP30.
For lava purple stained gels excitation/emission wavelengths were as follows 532 nm/560 nm LP.
6.2.2 Reference to gel matrix
There is only one gel in this document.
7. Image
7.1.1 Image name (or id)
CCB5_1 (format: BMP)
7.1.2 Dimensions
Width: 2478 px
Height: 1908 px
7.1.3 Resolution
96 dpi
7.1.4 Bit-depth
32-bit (TrueColor)7.1.5 Image location
CCB5_1.bmp7.1.6 Standard image orientation
Yes
7. Image
7.1.1 Image name (or id)
CCB90_1 (format: BMP)
7.1.2 Dimensions
Width: 2478 px
Height: 1908 px
7.1.3 Resolution
96 dpi
7.1.4 Bit-depth
32-bit (TrueColor)7.1.5 Image location
CCB90_1.bmp7.1.6 Standard image orientation
Yes
7. Image
7.1.1 Image name (or id)
STD4 (format: BMP)
7.1.2 Dimensions
Width: 2478 px
Height: 1908 px
7.1.3 Resolution
96 dpi
7.1.4 Bit-depth
32-bit (TrueColor)7.1.5 Image location
Internal Std gel 10.bmp7.1.6 Standard image orientation
Yes