Name: TCA-acetone-phenol_3 Description: TCA-acetone-phenol extraction of Arabidopsis leaf proteome Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2007-04-30 1.1.2 Responsible person or role Affiliation: Agricultural and Plant Biochemistry and Proteomics Research Group, Dept. Biochemistry and Molecular Biology, University of Córdoba, Spain (i) Name: Prof. Jesus V. Jorrin Novo (ii) Postal address: Dpto. Bioquimica y Biologia Molecular, Universidad de Cordoba, Campus de Rabanales, edificio Severo Ochoa, planta baja, 14014, Cordoba, Spain (iii) Email address: bf1jonoj@uco.es 1.1.3 Electrophoresis type Two-Dimensional electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: Arabidopsis thaliana leaf proteins extracted with a TCA-acetone-phenol method 2. Sample type: Test sample 2.1.2 Loading buffer 1. Urea 9 M, CHAPS 4 %, DTT 20 mM, Ampholytes 2 %, bromophenol blue 0.005 % 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: BioRad Model: IPG strip 17 cm pH 5-8 Model number: 163-2011 Batch number: unknown 3.2.3 Physical dimensions X: 17.1 cm Y: 0.33 cm Z: 0.05 cm 3.2.4 Physiochemical property range and distribution linear pH 5 - 8 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: Arabidopsis thaliana leaf proteins extracted with a TCA-acetone-phenol method * Volume of sample: 100 µg * Loading buffer: Urea 9 M, CHAPS 4 %, DTT 20 mM, Ampholytes 2 %, bromophenol blue 0.005 % * Volume of loading buffer: 300 µL Loading method: rehydration loading. 3.3 Protocol 3.3.1 Buffers No buffer. 3.3.2 Electrophoresis conditions Running temperature: 20 °C Gradient: 0-250 V, 20 min Gradient: 250-10000 V, 2.5 h Hold: 10000 V, 3.5 h 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name reduction 4.1.2 Inter dimension buffer Tris-HCl 375 mM pH 8.8, urea 6 M, SDS 2%, glycerol 20 %, DTT 2% 4.1.3 Additional reagents No additional reagent. 4.1.4 Equipment 4.1.5 Protocol Temperature: 25 °C. Duration: 15 min. Protocol: Remove the mineral oil from the IPG strips by placing them (gel side up) onto a piece of dry filter paper and blotting with a second piece of wet filter paper. Add 4 ml reduction buffer to laned tray, using one channel per IPG strip. Transfer the blotted IPG strips (gel side up) into the tray. Place the tray on an orbital shaker and gently shake for 15 m. 4.1.1 Step name alkylation 4.1.2 Inter dimension buffer Tris-HCl 375 mM pH 8.8, urea 6 M, SDS 2%, glycerol 20 %, DTT 2,5 % 4.1.3 Additional reagents No additional reagent. 4.1.4 Equipment 4.1.5 Protocol Temperature: 25 °C. Duration: 15 min. Protocol: Discard the used reduction buffer by carefully decanting the liquid from the tray. Remove the last few drops of reduction buffer. Add the indicated volume alkylation buffer to each IPG strip. Return the tray to 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 receipe: 34 % of destilated water 40 % of a 30 % acrylamide/Bis solution 25 % of 1.5 M Tris-HCl pH 8.8 1 % of a 10 % SDS solution 0.5 % of a 10% APS solution 0.05% of TEMED 3.2.3 Physical dimensions X: 20 cm Y: 20 cm Z: 0.1 cm 3.2.4 Physiochemical property range and distribution linear apparent molecular mass 100 - 12 kDa 3.2.5 Acrylamide concentration 12 % 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: IPG strip placing. 3.3 Protocol 3.3.1 Buffers 0.025 M Tris-HCl pH 8.3, 0.192 M glycine, 0.1 % SDS 3.3.2 Electrophoresis conditions Running temperature: 18 °C Hold: 600 mA, 18 h 5. Detection ------------ 5.1 Direct detection 5.1.1 Name of direct detection_process Fluorescent staining 5.1.2 Direct detection agents SyPro Ruby, Sigma 5.1.3 Additional reagents and buffers Fixation and destaining solution: 10 % methanol, 7 % acetic acid. 5.1.4 Equipment No specialised equipment. 5.1.5 Direct detection protocol Temperature: 25 °C. Duration: 18 h. Protocol: Put the gels in fixing solution during 30 min. Stain the gels in SyPro Ruby solution during 18 h, kept from light. Destain the gels with destaining solution during 30 min, kept from light. 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment fluorescent scanner 6.1.2 Name of equipment Manufacturer: BioRad Model: Molecular Imager FX Pro Plus multiImager System Model number: unknown 6.1.3 Software Manufacturer: BioRad Model: PDQuest Model number: 7.1 6.1.4 Calibration Yes (automatic) 6.1.5 Equipment specific parameters Visual inspection of the matching process 6.2 Acquisition Protocol 6.2.1 Image acquisition process 100x100 microns 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) TCA-acetone-phenol_3 (format: TIFF) 7.1.2 Dimensions Width: 1850 px Height: 1694 px 7.1.3 Resolution 100 µm/px 7.1.4 Bit-depth 8-bit (256 colors) 7.1.5 Image location TCA-acetone-phenol_3.tif Link: /download/23/cKVUX36T/ 7.1.6 Standard image orientation Yes