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. 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 Link: /download/585/BzunDYst/ 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 Link: /download/585/yosyNC9a/ 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 Link: /download/585/7nKr1wpE/ 7.1.6 Standard image orientation Yes