Name: Analysis of freezing stress modulated secretome of Hippophae rhamnoides Description: Twenty days old seabuckthorn seedlings were given -5 °C treatment and extracellular proteins were extracted. 250 µg of the proteins were loaded on 3-10 nonlinear IPG strip. Second dimension was carried out at 15% SDS-PAGE and the gel was silver stained. Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2010-04-01 1.1.2 Responsible person or role Affiliation: University of Delhi (i) Name: Dr. Renu Deswal (ii) Postal address: Molecular Plant Physiology and Proteomics Laboratory, Department of Botany, University of Delhi (iii) Email address: rdeswal@botany.du.ac.in 1.1.3 Electrophoresis type Two-Dimensional electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: Freezing stress modulated extracellular (apoplastic) protein from shoot of Hippophae rhamnoides seedlings 2. Sample type: Test sample 3. URL: http://pubs.acs.org/doi/abs/10.1021/pr200944z?mi=qryllt&af=R&pageSize=20&searchText=ATP Link: http://pubs.acs.org/doi/abs/10.1021/pr200944z?mi=qryllt&af=R&pageSize=20&searchText=ATP 2.1.2 Loading buffer 1. 8 M urea, 2 M thiourea, 2% w/v CHAPS, 50 mM DTT, 0.5% v/v IPG buffer, 2% Triton X-100 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: G E Healthcare Life Sciences Model: IPG Strip 3-10 non linear Model number: 17-6001-14 Batch number: Unknown 3.2.3 Physical dimensions X: 130 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: Freezing stress modulated extracellular (apoplastic) protein from shoot of Hippophae rhamnoides seedlings * Volume of sample: 250 µg * Loading buffer: 8 M urea, 2 M thiourea, 2% w/v CHAPS, 50 mM DTT, 0.5% v/v IPG buffer, 2% Triton X-100 and a trace of bromophenol blue * Volume of loading buffer: 250 µL Loading method: rehydration loading. 3.3 Protocol 3.3.1 Buffers No buffer. 3.3.2 Electrophoresis conditions Running temperature: 20 °C Hold: 500 V, 4 h Gradient: 500-1000 V, 1.30 h Gradient: 1000-8000 V, 2.3 h Hold: 8000 V, 0.3 h 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name equilibration 4.1.2 Inter dimension buffer 6 M urea, 30% v/v glycerol, 2% w/v SDS, 50 mM Tris-HCl, pH 8.8 4.1.3 Additional reagents Reduction-10 mg/mL DTT Alkylation-25 mg/mL iodoacetamide 4.1.4 Equipment Manufacturer: No Specialised equipment was used Model: No Specialised equipment was used Model number: No Specialised equipment was used 4.1.5 Protocol Temperature: 20 °C. Duration: 20 min. Protocol: Strip was first equilibrated with 5 mL equilibration buffer A containing 10 mg/mL DTT for 20 min and then replaced with equilibration buffer B containing 25 mg/mL iodoacetamide for next 20 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: CSH Protocols,doi:10.1101pdb.prot4540, page(s) doi:10.1101pdb.prot4540 (2006). URL: http://vetbiotech.um.ac.ir/parameters/vetbiotech/filemanager/new_admin/electrophorsis/SDS-Polyacrylamide%20Gel%20Electrophoresis%20of%20Proteins.pdf Link: http://vetbiotech.um.ac.ir/parameters/vetbiotech/filemanager/new_admin/electrophorsis/SDS-Polyacrylamide%20Gel%20Electrophoresis%20of%20Proteins.pdf 3.2.3 Physical dimensions X: 180 mm Y: 160 mm Z: 1 mm 3.2.4 Physiochemical property range and distribution logarithmic apparent molecular mass 14.2 - 97 kDa 3.2.5 Acrylamide concentration 15 % 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: Bisacrylamide Ratio: 30:0.8 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 0.1% SDS 3.3.2 Electrophoresis conditions Running temperature: room temperature (no cooling device). Hold: 10 mA, 0.3 h Hold: 20 mA, 6 h 5. Detection ------------ 5.1 Direct detection 5.1.1 Name of direct detection_process Silver staining 5.1.2 Direct detection agents Silver nitrate 5.1.3 Additional reagents and buffers 45% methnol and 10% acetic acid (fixing solution), 20 mg/100 mL sodium thiosulphate (sensitizer), 2 g/100 mL (developer) and 10% acetic acid (Stopper) 5.1.4 Equipment Manufacturer: No Specialised equipment was used Model: No Specialised equipment was used Model number: No Specialised equipment was used 5.1.5 Direct detection protocol Temperature: 4 °C. Duration: 10 min. Detection is described in the following reference protocol: Citation: Methods in enzymology,96, page(s) 230-239 (1983). URL: not provided. 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment camera 6.1.2 Name of equipment Manufacturer: AlphaImager gel documentation system Model: Alpha imager Model number: AlphaImager HP System 6.1.3 Software Manufacturer: AlphaImager gel documentation system Model: Alpha View Software Model number: Alpha View Software 6.1.4 Calibration Yes (automatic) 6.1.5 Equipment specific parameters Default (vendor) parameters. Configuration file: Freezing stress modulated extracellular proteins of Hippophae rhamnoides Link: /download/580/8sTpfnID/ 6.2 Acquisition Protocol 6.2.1 Image acquisition process Image was acquired 2 sec 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) HRHAMNOIDES_SEEDLINGS (ECP)_3-10 (format: TIFF) 7.1.2 Dimensions Width: 1153 px Height: 1000 px 7.1.3 Resolution 102 ppi 7.1.4 Bit-depth 8-bit (256 colors) 7.1.5 Image location http://pubs.acs.org/doi/abs/10.1021/pr200944z?mi=qryllt&af=R&pageSize=20&searchText=ATP Link: http://pubs.acs.org/doi/abs/10.1021/pr200944z?mi=qryllt&af=R&pageSize=20&searchText=ATP 7.1.6 Standard image orientation Yes