Name: Test gel Description: This is the protocol example for a 2-DE gel Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2004-09-04 1.1.2 Responsible person or role Affiliation: SIB (i) Name: Xavier Robin (ii) Postal address: 1, rue Michel Servet, CH-1211 Genève 4, Switzerland (iii) Email address: robin0@etu.unige.ch 1.1.3 Electrophoresis type Two-Dimensional electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: Colon epithelial cell (CEC) 2. Sample type: Test sample 3. URL: http://www.expasy.org/ch2d/protocols/protocols.fm1.html#998547 Link: http://www.expasy.org/ch2d/protocols/protocols.fm1.html#998547 2.1.2 Loading buffer 1. Urea (8 M), CHAPS (4 % w/v), DTE (65 mM), Tris (40 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: GE Healthcare 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 linear pH 3.5 - 10 3.2.5 Acrylamide concentration 4 % 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: Bisacrylamide Ratio: 32.3:1 3.2.7 Additional substances in gel No additional substance 3.2.8 Gel lane 1 3.2.9 Sample application Lane 1 * Sample: Colon epithelial cell (CEC) * Volume of sample: 106 µg * Loading buffer: Urea (8 M), CHAPS (4 % w/v), DTE (65 mM), Tris (40 mM) and a trace of bromophenol blue. * Volume of loading buffer: 100 µL * Volume of mixture loaded in the lane: 100 µL Loading method: cup loading. Additional comment: Rehydration : Hydration was performed overnight in the Pharmacia reswelling cassette with 25 ml of a solution containing urea (8 M), CHAPS (2% w/v), DTE (10 mM), Resolyte pH 3.5-10 (2% v/v) and a trace of Bromophenol Blue. After placing rehydrated 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. 3.3 Protocol 3.3.1 Buffers No buffer. 3.3.2 Electrophoresis conditions Running temperature: 8-12 °C Gradient: 300-3500 V, 3 h Hold: 3500 V, 3 h Hold: 5000 V, 8 h 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name equilibration (resolubilization and reduction) 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 Model: Strip tray Model number: unknown 4.1.5 Protocol Duration: 12 min. Protocol: The strips were equilibrated within the strip tray with 100 ml of the solution described above (4.1.2, 4.1.3) for 12 min. 4.1.1 Step name blocking 4.1.2 Inter dimension buffer Tris-HCl (50mM) pH 6.8. 4.1.3 Additional reagents Urea (6 M), glycerol (30% v/v), SDS (2% w/v), iodoacetamide (2.5% w/v) and a trace of Bromophenol blue. 4.1.4 Equipment Manufacturer: Pharmacia Model: Strip tray Model number: unknown 4.1.5 Protocol Duration: 5 min. Protocol: -SH groups were subsequently blocked with 100 ml of the solution described above (4.1.2, 4.1.3) 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: Anal. Biochem.,173, page(s) 412-423 (1988). URL: not provided. 3.2.3 Physical dimensions X: 160 mm Y: 200 mm Z: 1.5 mm 3.2.4 Physiochemical property range and distribution linear apparent molecular mass 20 - 200 kDa 3.2.5 Acrylamide concentration 9 - 16 % (linear) 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: PDA Ratio: 37.5:1 3.2.7 Additional substances in gel TEMED 0.05% Sodium thiosulfate 5mM APS 0.1% 3.2.8 Gel lane 1 3.2.9 Sample application Loading method: gel loading. 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 70°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, 4 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 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. 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment camera 6.1.2 Name of equipment Manufacturer: Molecular Dynamics Model: Laser Densitometer Model number: unknown 6.1.3 Software Manufacturer: GE Healthcare Model: ImageQuant Model number: unknown 6.1.4 Calibration Yes (automatic) 6.1.5 Equipment specific parameters Parameters used are now unknown Configuration file: Configuration file for the software Link: /download/1/2tYUsBlX/ 6.2 Acquisition Protocol 6.2.1 Image acquisition process Protocol used is now 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) CEC_HUMAN.mel (format: TIFF) 7.1.2 Dimensions Width: 5000 px Height: 4000 px 7.1.3 Resolution 20 px/mm 7.1.4 Bit-depth 12-bit 7.1.5 Image location CEC_HUMAN.mel Link: /download/1/omKBWIR9/ 7.1.6 Standard image orientation Yes