Name: Two-dimensional gel of Spartina alterniflora Description: Heat-stable proteins (95C for 40 min) were extracted from Spartina alterniflora, and separated on two-dimensinoal gels (pI 3-10 NL, 24 cm IPG). Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2011-01-05 1.1.2 Responsible person or role Affiliation: Louisiana State University, AgCenter (i) Name: Wang, Yi (ii) Postal address: Department of Plant Pathology and Crop Physiology 302 Life Sciences Building Louisiana State University Baton Rouge, LA 70803 (iii) Email address: ywang17@tigers.lsu.edu 1.1.3 Electrophoresis type Two-Dimensional electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: Heat stable proteins from Spartina alterniflora seeds 2. Sample type: Test sample 2.1.2 Loading buffer 1. 7M urea, 2M thiourea, 4% CHAPS (w/v) and 20mM dithiothreitol (DTT), 2% (w/v) IPG buffer NL 3-10. Rehydration buffer: 7 M urea, 2 M thiourea, 0.001% (w/v) bromophenol, 2% (w/v) CHAPS, 20 mM DTT and 0.5% (w/v) IPG buffer NL 3-10. 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: Immobiline DryStrip pH 3-10 NL, 24 cm Model number: 17-6002-45 Batch number: 10057246 3.2.3 Physical dimensions X: 240 mm Y: 3 mm Z: 0.5 mm 3.2.4 Physiochemical property range and distribution NL 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 5 3.2.9 Sample application Lane 1 * Sample: Heat stable proteins from Spartina alterniflora seeds * Volume of sample: 1000 µg * Loading buffer: 7M urea, 2M thiourea, 4% CHAPS (w/v) and 20mM dithiothreitol (DTT), 2% (w/v) IPG buffer NL 3-10. Rehydration buffer: 7 M urea, 2 M thiourea, 0.001% (w/v) bromophenol, 2% (w/v) CHAPS, 20 mM DTT and 0.5% (w/v) IPG buffer NL 3-10. * Volume of loading buffer: 30 µL Lane 2 * Sample: Heat stable proteins from Spartina alterniflora seeds * Volume of sample: 1000 µg * Loading buffer: 7M urea, 2M thiourea, 4% CHAPS (w/v) and 20mM dithiothreitol (DTT), 2% (w/v) IPG buffer NL 3-10. Rehydration buffer: 7 M urea, 2 M thiourea, 0.001% (w/v) bromophenol, 2% (w/v) CHAPS, 20 mM DTT and 0.5% (w/v) IPG buffer NL 3-10. * Volume of loading buffer: 30 µL Lane 3 * Sample: Heat stable proteins from Spartina alterniflora seeds * Volume of sample: 1000 µg * Loading buffer: 7M urea, 2M thiourea, 4% CHAPS (w/v) and 20mM dithiothreitol (DTT), 2% (w/v) IPG buffer NL 3-10. Rehydration buffer: 7 M urea, 2 M thiourea, 0.001% (w/v) bromophenol, 2% (w/v) CHAPS, 20 mM DTT and 0.5% (w/v) IPG buffer NL 3-10. * Volume of loading buffer: 30 µL Lane 4 * Sample: Heat stable proteins from Spartina alterniflora seeds * Volume of sample: 1000 µg * Loading buffer: 7M urea, 2M thiourea, 4% CHAPS (w/v) and 20mM dithiothreitol (DTT), 2% (w/v) IPG buffer NL 3-10. Rehydration buffer: 7 M urea, 2 M thiourea, 0.001% (w/v) bromophenol, 2% (w/v) CHAPS, 20 mM DTT and 0.5% (w/v) IPG buffer NL 3-10. * Volume of loading buffer: 30 µL Lane 5 * Sample: Heat stable proteins from Spartina alterniflora seeds * Volume of sample: 1000 µg * Loading buffer: 7M urea, 2M thiourea, 4% CHAPS (w/v) and 20mM dithiothreitol (DTT), 2% (w/v) IPG buffer NL 3-10. Rehydration buffer: 7 M urea, 2 M thiourea, 0.001% (w/v) bromophenol, 2% (w/v) CHAPS, 20 mM DTT and 0.5% (w/v) IPG buffer NL 3-10. * Volume of loading buffer: 30 µL Loading method: rehydration loading. Additional comment: Immobiline DryStrip IPGbox rehydration for 12 hours. 3.3 Protocol 3.3.1 Buffers No buffer. 3.3.2 Electrophoresis conditions Running temperature: 20 °C Hold: 500 V, 2 h Hold: 1000 V, 2 h Gradient: 1000-8000 V, 2 h Hold: 8000 V, 3 h Gradient: 8000-10000 V, 1 h Hold: 10000 V, 1 h 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name reduction 4.1.2 Inter dimension buffer 50 mM Tris-HCl (pH 8.8), 6 M urea, 30% glycerol (v/v), 2% SDS (w/v), 0.002% (w/v) bromophenol 4.1.3 Additional reagents 0.5% DTT (w/v) 4.1.4 Equipment Manufacturer: unknown Model: Equilibrium tray 12 wells Model number: unknown 4.1.5 Protocol Temperature: 22 °C. Duration: 30 min. Protocol: Strips immersed in individual wells of a rehydration tray and kept shaking for 30 minutes. 4.1.1 Step name alkylation 4.1.2 Inter dimension buffer 50 mM Tris-HCl (pH 8.8), 6 M urea, 30% glycerol (v/v), 2% SDS (w/v), 0.002% (w/v) bromophenol 4.1.3 Additional reagents 1.25% Iodoacetamide (w/v) 4.1.4 Equipment Manufacturer: unknown Model: Equilibrium tray 12 wells Model number: unknown 4.1.5 Protocol Temperature: 22 °C. Duration: 40 min. Protocol: Strips immersed in individual wells of a rehydration tray and kept shaking for 40 minutes. 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: A recipe to prepare 1200 ml of 12.5% polyacrylamide gels [150.0 g acrylamide, 4.032 g bis-acrylamide, 1.5 M Tris-HCl pH 8.8, 10% SDS (w/v), 10% APS (w/v) and 340 µl TEMED] (the stirred gel solution was degassed under vacuum for 20 minutes, and APS and TEMED were not added until gels were ready to pour) was used to make 12 gels. 3.2.3 Physical dimensions X: 260 mm Y: 200 mm Z: 1 mm 3.2.4 Physiochemical property range and distribution logarithmic apparent molecular mass 100 - 6 kDa 3.2.5 Acrylamide concentration 12.5 % 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: Bisacrylamide Ratio: 37.2:1 3.2.7 Additional substances in gel No additional substance 3.2.8 Gel lane 5 3.2.9 Sample application Loading method: IPG strip. Additional comment: IPG strip was placed on top of the slab gel and held in place with Agarose Sealing Solution (0.5% agarose in 1x SDS Electrophoresis Buffer). 3.3 Protocol 3.3.1 Buffers 1x SDS running buffer: 2.5 mM Tris-base pH 8.3, 19.2 mM glycine and 0.01 % (w/v) SDS. 2x SDS topping buffer: 5 mM Tris-base pH 8, 38.4 mM glycine and 0.02% (w/v) SDS. 3.3.2 Electrophoresis conditions Running temperature: 18 °C Hold: 40 V, 1 h Hold: 110 V, 13 h 5. Detection ------------ 5.1 Direct detection 5.1.1 Name of direct detection_process Colloidal Coomassie Blue G-250 staining (Blue silver staining) 5.1.2 Direct detection agents 2% H3PO4, 10% (NH4)2SO4, 20% methanol and 0.1% Coomassie G-250. The preparation of the staining recipe (10 L) was by sequentially addition as follows: 235 ml of phosphoric acid (85%, w/v) was added to 2 L of distilled water; 1 kg of ammonium sulfate powder was added to 4 L of distilled water. After they were fully dissolved, phosphoric acid and ammonium sulfate solutions were combined and poured into the staining tank together with 1.5 L of distilled water. Ten grams of Coomassie Brilliant Blue G-250 were dissolved in 500 ml distilled water, and then added to staining tank. Two liters of methanol were not added to staining tank until the gel staining began. 5.1.3 Additional reagents and buffers No additional reagents or buffer 5.1.4 Equipment Manufacturer: Bio-Rad Model: Dodeca Stainer Model number: #165-3401 5.1.5 Direct detection protocol Temperature: 22 °C. Duration: 4 d. Protocol: The staining took place for 2-3 days, and gels were de-stained with distilled water until the background was clear (8-10 hours in most cases). 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment Professional scanner 6.1.2 Name of equipment Manufacturer: UMAX Model: UTA Model number: 1100 6.1.3 Software Manufacturer: UMAX Model: Magicscan Model number: 4.5 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 images were acquired in 16 bit grayscale with a resolution of 300 dpi and were saved in tiff format. No filters or image processing tools were used during acquisition. 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) SA Gel #1 - #5 (format: TIFF) 7.1.2 Dimensions Width: 2196 px Height: 2954 px 7.1.3 Resolution 300 dpi 7.1.4 Bit-depth 16-bit (HighColor) 7.1.5 Image location sa #1.tif Link: /download/568/hpbgUIGv/ 7.1.6 Standard image orientation Yes