Name: NCB0_vs_NCB15_repl1

Description: This gel is composed of a nuclear fraction of a Cy-5 labeled negative control (biological replicate 1), a nuclear fraction of a Cy-3 labeled 15min 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)

    1. Sample name: NCB0_1(Cy5)+NCB15_1(Cy3)+Internal standard (Cy2)
    2. Sample type: Test sample

2.1.2 Loading buffer

  1. 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

1

3.2.9 Sample application

Lane 1

  • Sample: NCB0_1(Cy5)+NCB15_1(Cy3)+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

1

3.2.9 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 scanner

6.1.2 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)

NCB0_1 (format: BMP)

7.1.2 Dimensions

Width: 2491 px

Height: 1987 px

7.1.3 Resolution

96 dpi

7.1.4 Bit-depth

32-bit (TrueColor)

7.1.5 Image location

NCB0_1.bmp

7.1.6 Standard image orientation

Yes

7. Image

7.1.1 Image name (or id)

NCB15_1 (format: BMP)

7.1.2 Dimensions

Width: 1987 px

Height: 2491 px

7.1.3 Resolution

96 dpi

7.1.4 Bit-depth

32-bit (TrueColor)

7.1.5 Image location

NCB15_1.bmp

7.1.6 Standard image orientation

Yes

7. Image

7.1.1 Image name (or id)

STD7 (format: BMP)

7.1.2 Dimensions

Width: 2491 px

Height: 1987 px

7.1.3 Resolution

96 dpi

7.1.4 Bit-depth

32-bit (TrueColor)

7.1.5 Image location

Internal Std gel 19.bmp

7.1.6 Standard image orientation

Yes

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