Name: Human macrophage like cell line (Analytical 2-DE) Description: Human macrophage like cell line (U937) - Analytical 2-DE Reference: Sanchez J.-C., Appel R.D., Golaz O., Pasquali C., Ravier F., Bairoch A., Hochstrasser D.F. Inside SWISS-2DPAGE database. Electrophoresis 1995, 16, 1131-1151. ( doi:10.1002/elps.11501601190 Link: http://dx.doi.org/10.1002/elps.11501601190) Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 1994-08-01 1.1.2 Responsible person or role Affiliation: Swiss Institute of Bioinformatics (i) Name: Christine Hoogland (ii) Postal address: Swiss Institute of Bioinformatics CMU - 1, rue Michel Servet 1211 Geneva 4 (iii) Email address: christine.hoogland@isb-sib.ch 1.1.3 Electrophoresis type Two-Dimensional electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: Human macrophage like cell line 2. Sample type: Test sample 3. URL: http://www.expasy.org/ch2d/protocols/protocols.fm1.html Link: http://www.expasy.org/ch2d/protocols/protocols.fm1.html 2.1.2 Loading buffer 1. urea (8 M), CHAPS (4% w/v), Tris (40 mM), DTE (65 mM) and a trace of bromophenol blue 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: Pharmacia-Hoeffer Model: IPGs NL pH 3.5-10 Model number: 17-1235-01 Batch number: unknown 3.2.3 Physical dimensions X: 180 mm Y: 3 mm Z: 1.5 mm 3.2.4 Physiochemical property range and distribution non-linear pH 3.5 - 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: Human macrophage like cell line * Volume of sample: 0.8 µg * Loading buffer: urea (8 M), CHAPS (4% w/v), Tris (40 mM), DTE (65 mM) and a trace of bromophenol blue * Volume of loading buffer: 60 µL * Volume of mixture loaded in the lane: 60 µL Loading method: well loading. Additional comment: When the rehydration cassette had been thoroughly emptied and opened, the strips were transferred to the Pharmacia strip tray. After placing IPG strips, humid electrode wicks, electrodes and sample cups in position, the strips and cups were covered with low viscosity paraffin oil. Samples were applied at the cathodic end of the IPG strips in a slow and continuous manner, without touching the gels [6]. 3.3 Protocol 3.3.1 Buffers No buffer. 3.3.2 Electrophoresis conditions Running temperature: room temperature (no cooling device). Gradient: 300-3500 V, 3 h Hold: 3500 V, 3 h Hold: 5000 V, 18 h 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name equilibration 4.1.2 Inter dimension buffer Tris-HCL (50mM) pH 8.4 4.1.3 Additional reagents Urea (6 M), glycerol (30% v/v), SDS (2% w/v) and DTE (2% w/v) 4.1.4 Equipment Manufacturer: Pharmacia-Hoeffer Model: Immobiline strip tray Model number: unknown 4.1.5 Protocol Duration: 12 min. Protocol: 100ml of this solution for 12min 4.1.1 Step name SH_blocking 4.1.2 Inter dimension buffer Tris-HCl (50 mM) pH 6.8, urea (6 M), glycerol (30% v/v), SDS (2% w/v), iodoacetamide (2.5% w/v) and a trace of Bromophenol Blue 4.1.3 Additional reagents No additional reagent. 4.1.4 Equipment Manufacturer: Pharmacia-Hoeffer Model: Immobiline strip tray Model number: unknown 4.1.5 Protocol Duration: 5 min. Protocol: 100 ml of solution for 5 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 reference protocol: Citation: not provided. URL: http://www.expasy.org/ch2d/protocols/protocols.fm3.html Link: http://www.expasy.org/ch2d/protocols/protocols.fm3.html 3.2.3 Physical dimensions X: 160 mm Y: 200 mm Z: 1.5 mm 3.2.4 Physiochemical property range and distribution logarithmic apparent molecular mass 10 - 200 kDa 3.2.5 Acrylamide concentration 9-16 % (sigmoidal) 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: PDA Ratio: 37.5:1 3.2.7 Additional substances in gel Sodium thiosulfate 5mM TEMED 0.05% APS 0.1% 3.2.8 Gel lane 1 3.2.9 Sample application Loading method: IPG_transfer. Additional comment: After the equilibration, the IPG gel strips were cut to size. Six mm were removed from the anodic end and 14 mm from the cathodic end. The second dimension gels were overlayered with a solution containing agarose (0.5% w/v) and Tris-glycine-SDS (25 mM-198 mM-0.1% w/v) pH 8.3 heated at about 70o C and the IPG gel strips were immediately loaded through it 3.3 Protocol 3.3.1 Buffers Running buffer: Tris-glycine-SDS (25 mM-198 mM-0.1% w/v) pH 8.3 Leading buffer: Tris-HCl (0.375 M) pH 8.8 3.3.2 Electrophoresis conditions Running temperature: 8-12 °C Hold: 40 mA, 5 h 5. Detection ------------ 5.1 Direct detection 5.1.1 Name of direct detection_process Ammoniacal silver staining 5.1.2 Direct detection agents 6g silver 5.1.3 Additional reagents and buffers Solution 1: ethanol: acetic acid: water (40: 10: 50) Solution 2: ethanol: acetic acid: water (5: 5: 90) Solution 3: glutaraldehyde (1%) and sodium acetate (0.5 M) Solution 4: 2,7 naphtalene-disulfonic acid solution (0.05% w/v) Solution 5: 6 g of silver nitrate were dissolved in 30 ml of deionized water, which was slowly mixed into a solution containing 160 ml of water, 10 ml of concentrated ammonia (25%) and 1.5 ml of sodium hydroxide (10 N). A transient brown precipitate might form. After it cleared, water was added to give the final volume. Solution 6: citric acid (0.01% w/v) and formaldehyde (0.1% v/v) Solution 7: Tris (5% w/v) and acetic acid (2% v/v) 5.1.4 Equipment No specialised equipment. 5.1.5 Direct detection protocol Duration: 5 h. Protocol: 1. At the end of the second dimension run, the gels were removed from the glass plates and washed in deionized water for 5 min. 2. Soaked in Solution 1 for 1 hour. 3. Soaked in Solution 2 for 2 hours or overnight. 4. Washed in deionized water for 5 min. 5. Soaked in Solution 3 for 30 min. 6. Washed 3 times in deionized water for 10 min. 7. In order to obtain homogeneous dark brown staining of proteins, gels were soaked twice in 750 ml Solution 4 for 30 min. 8. Rinsed 4 times in deionized water for 15 min. 9. Gels were stained in a freshly made Solution 5 for 30 minutes. 10. After staining, the gels were washed 4 times in deionized water for 4 min. 11. The images were developed in Solution 6 for 5 to 10 min. 12. When a slight background stain appeared, development was stopped with Solution 7. Detection is described in the following reference protocol: Citation: not provided. URL: http://www.expasy.org/ch2d/protocols/protocols.fm4.html Link: http://www.expasy.org/ch2d/protocols/protocols.fm4.html 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment laser scanner 6.1.2 Name of equipment Manufacturer: Molecular Dynamics, CA Model: Laser Densitometer (4000 x 5000 pixels; 12 bits/pixel) Model number: unknown 6.1.3 Software Manufacturer: Molecular Dynamics, CA Model: ImageQuant Model number: 3.0 6.1.4 Calibration Yes (automatic) 6.1.5 Equipment specific parameters unknown 6.2 Acquisition Protocol 6.2.1 Image acquisition process unknown 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) U937_HUMAN.mel (format: TIFF) 7.1.2 Dimensions Width: 963 px Height: 1115 px 7.1.3 Resolution 300 dpi 7.1.4 Bit-depth 16-bit (HighColor) 7.1.5 Image location ftp://ftp.expasy.org/databases/swiss-2dpage/MASTERS/U937_HUMAN.mel Link: ftp://ftp.expasy.org/databases/swiss-2dpage/MASTERS/U937_HUMAN.mel 7.1.6 Standard image orientation Yes