Name: Analysis of leaf proteome of Vigna mungo (VM4) control Description: Analysis of leaf proteome of Vigna mungo (VM4) control Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2009-08-25 1.1.2 Responsible person or role Affiliation: Bose Institute (i) Name: Prof Amita Pal (ii) Postal address: Division of Plant Biology, Bose Institute, Kolkata 700054, West Bengal, India (iii) Email address: amita@bic.boseinst.ernet.in 1.1.3 Electrophoresis type Two-Dimensional electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: VM4 Control 2. Sample type: Control sample 2.1.2 Loading buffer 1. 8 M Urea, 2% CHAPS, 50 mM DTT, 0.2 % Bio-lyte 3/10 ampholyte and 0.001 % 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: Bio-rad Model: ReadyStripTM IPG Strip pH 3 - 10 Non-linear 17 cm strips Model number: 163-2009 Batch number: Unknown 3.2.3 Physical dimensions X: 178 mm Y: 3.3 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: 32.3:1 3.2.7 Additional substances in gel No additional substance 3.2.8 Gel lane 1 3.2.9 Sample application Lane 1 * Sample: VM4 Control * Volume of sample: 400 µg * Loading buffer: 8 M Urea, 2% CHAPS, 50 mM DTT, 0.2 % Bio-lyte 3/10 ampholyte and 0.001 % Bromophenol Blue * Volume of loading buffer: 300 µL Loading method: Passive rehydration. 3.3 Protocol 3.3.1 Buffers No buffer. 3.3.2 Electrophoresis conditions Running temperature: 20 °C Gradient: 0-250 V, 15 min Gradient: 250-10000 V, 2.5 h Hold: 10000 V, 6 h 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name reduction 4.1.2 Inter dimension buffer 20% v/v glycerol, 0.375 M Tris–HCl, pH 8.8, 6 M Urea, 2% (w/v) SDS, 130 mM DTT 4.1.3 Additional reagents No additional reagent. 4.1.4 Equipment 4.1.5 Protocol Temperature: 25 °C. Duration: 15 min. Protocol: The mineral oil was removed from the IPG strips by placing them (gel side up) onto a piece of dry filter paper and blotted with a second piece of wet filter paper. 5 ml reduction buffer was added to the channeled tray. IPG strips (gel side up) were transfer into the tray. The tray was placed on an orbital shaker and gently shakes for 15 m. 4.1.1 Step name alkylation 4.1.2 Inter dimension buffer 20% v/v glycerol, 0.375 M Tris–HCl, pH 8.8, 6 M Urea, 2% (w/v) SDS, 135 mM iodoacetamide 4.1.3 Additional reagents No additional reagent. 4.1.4 Equipment 4.1.5 Protocol Temperature: 25 °C. Duration: 15 min. Protocol: The reduction buffer was removed carefully by decanting the liquid from the tray. 5 ml alkylation buffer was added to each IPG strip. The tray was kept on the orbital shaker for 15 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: Nature,227, page(s) 680-685 (1970). URL: not provided. 3.2.3 Physical dimensions X: 200 mm Y: 180 mm Z: 1.5 mm 3.2.4 Physiochemical property range and distribution linear apparent molecular mass 70 - 10 kDa 3.2.5 Acrylamide concentration 1 % 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: Bisacrylamide Ratio: 29:1 3.2.7 Additional substances in gel No additional substance 3.2.8 Gel lane 1 3.2.9 Sample application Loading method: IPG strip transfer. 3.3 Protocol 3.3.1 Buffers 25 mM Tris, 192 mM glycine and 0.1 % SDS 3.3.2 Electrophoresis conditions Running temperature: 20 °C Hold: 200 V, 7.5 h 5. Detection ------------ 5.1 Direct detection 5.1.1 Name of direct detection_process Coomassie blue staining 5.1.2 Direct detection agents Coomassie G-250 stain 5.1.3 Additional reagents and buffers Destaining solution: 20 % methanol, 10 % acetic acid. 5.1.4 Equipment No specialised equipment. 5.1.5 Direct detection protocol Temperature: 25 °C. Duration: 12 h. Protocol: The gels were put into staining solution for 12 h. Destain the gels with destaining solution 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment VersaDoc 6.1.2 Name of equipment Manufacturer: Bio-rad Model: VersaDoc Imaging System Model number: Unknown 6.1.3 Software Manufacturer: Bio-rad Model: 8.0.1 Model number: Unknown 6.1.4 Calibration Yes (automatic) 6.1.5 Equipment specific parameters Default (vendor) parameters. 6.2 Acquisition Protocol 6.2.1 Image acquisition process The gel image was acquired on VersaDoc Imaging system using Qunatity One (Bio-rad) software with 1 s exposure time. 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) VM4 Control (format: TIFF) 7.1.2 Dimensions Width: 1051 px Height: 1004 px 7.1.3 Resolution 254 ppi 7.1.4 Bit-depth 8-bit (256 colors) 7.1.5 Image location VM4 Control.tif Link: /download/329/7RrR4P5c/ 7.1.6 Standard image orientation Yes