Name: Lactobacillus acidophilus NCFM reference proteome (pI 6-11) Description: reference proteome of whole cell extract of Lactobacillus acidophilus NCFM Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2009-09-01 1.1.2 Responsible person or role Affiliation: Technical University of Denmark (i) Name: Susanne Jacobsen (ii) Postal address: Department of Systems Biology Enzyme and Protein Chemistry Technical University of Denmark Søltofts Plads Building 224, room 208 2800 Kgs. Lyngby Denmark (iii) Email address: sja@bio.dtu.dk 1.1.3 Electrophoresis type Two-Dimensional electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: Lactobacillus acidophilus NCFM reference proteome (pI 6-11) 2. Sample type: Standard 2.1.2 Loading buffer 1. 7 M urea, 2 M thiourea, 48 mM CHAPS, 200 mM bis(2-hydroxyethyl) disulfide (HED), 5% glycerol, 10% 2-propanol, and 2% pharmalyte pH 6−11 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 life sciences Model: Immobiline DryStrip pH 6-11, 18 cm Model number: 17-6001-97 Batch number: unknown 3.2.3 Physical dimensions X: 180 mm Y: 3 mm Z: 0.5 mm 3.2.4 Physiochemical property range and distribution linear pH 6 - 11 3.2.5 Acrylamide concentration 4 % 3.2.6 Acrylamide : Crosslinker ratio Crosslinker: Bisacrylamide Ratio: 4:3 3.2.7 Additional substances in gel No additional substance 3.2.8 Gel lane 1 3.2.9 Sample application Lane 1 * Sample: Lactobacillus acidophilus NCFM reference proteome (pI 6-11) * Volume of sample: 340 µL * Loading buffer: 7 M urea, 2 M thiourea, 48 mM CHAPS, 200 mM bis(2-hydroxyethyl) disulfide (HED), 5% glycerol, 10% 2-propanol, and 2% pharmalyte pH 6−11 * Volume of loading buffer: 100 µL Loading method: cup loading. Additional comment: IPG strips (pH 6−11, 18 cm; GE Life Sciences), were passively rehydrated (340 μL rehydration buffer) overnight (Immobiline DryStrip Reswelling Tray) at room temperature. Proteins (350 μg) from whole cell extract in 100 μL rehydration buffer were applied using cup-loading (Cup Loading Manifold; GE Life Sciences) according to the manufacturer’s instructions 3.3 Protocol 3.3.1 Buffers (7 M urea, 2 M thiourea, 48 mM CHAPS, 200 mM bis(2-hydroxyethyl) disulfide (HED), 5% glycerol, 10% 2-propanol, and 2% pharmalyte pH 6−11 (GE Life Sciences, Uppsala, Sweden)). 3.3.2 Electrophoresis conditions Running temperature: 20 °C Hold: 30 V, 10 min Hold: 150 V, 2 h Hold: 1000 V, 30 min Gradient: 1000-8000 V, 4 h Hold: 8000 V, 1.75 h 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name reduction 4.1.2 Inter dimension buffer 6 M urea, 30% glycerol, 50 mM Tris-HCl, pH 8.8, 2% SDS, 0.01% bromophenol blue supplemented with 1% DTT 4.1.3 Additional reagents 6 M urea, 30% glycerol, 50 mM Tris-HCl, pH 8.8, 2% SDS, 0.01% bromophenol blue) supplemented with 2.5% iodoacetamide 4.1.4 Equipment Manufacturer: Pyrex Model: Culture tubes Model number: 1636/36MP 4.1.5 Protocol Temperature: 23 °C. Duration: 15 min. Protocol: Strips were equilibrated in two steps of 15 min with 5 mL equilibration buffer (6 M urea, 30% glycerol, 50 mM Tris-HCl, pH 8.8, 2% SDS, 0.01% bromophenol blue) supplemented with 1% DTT and 2.5% iodoacetamide, respectively. 4.1.1 Step name reduction 4.1.2 Inter dimension buffer 6 M urea, 30% glycerol, 50 mM Tris-HCl, pH 8.8, 2% SDS, 0.01% bromophenol blue supplemented with 1% DTT 4.1.3 Additional reagents Alkylation buffer 6 M urea, 30% glycerol, 50 mM Tris-HCl, pH 8.8, 2% SDS, 0.01% bromophenol blue supplemented with 2.5% iodoacetamide 4.1.4 Equipment Manufacturer: Pyrex Model: Culture tubes Model number: 1636/36MP 4.1.5 Protocol Temperature: 23 °C. Duration: 15 min. Protocol: Strips were equilibrated in two steps of 15 min with 5 mL equilibration buffer (6 M urea, 30% glycerol, 50 mM Tris-HCl, pH 8.8, 2% SDS, 0.01% bromophenol blue) supplemented with 1% DTT and 2.5% iodoacetamide, respectively. 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: Reagents Quantity for 900 ml of a 12.5% gel Acrylamide/PAGE 40% (w/v)= 281.25 ml PlusOne Methylenebisacrylamide 2% (w/v)= 150.3 ml Tris (1.5 M, pH 8.8)= 225 ml 10% (w/v) SDS = 9.0 ml 10% (v/v) TEMED = 1.24 ml 10% (w/v) APS = 9.0 ml Make up to 900 ml with distilled water 3.2.3 Physical dimensions X: 27 cm Y: 21 cm Z: 0.015 cm 3.2.4 Physiochemical property range and distribution logarithmic apparent molecular mass 200 - 3 kDa 3.2.5 Acrylamide concentration 12.5 % 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: manual. Additional comment: 1. Briefly rinse the Immobiline DryStrips by submerging them in a measuring cylinder containing SDS electrophoresis running buffer for Ettan DALT. 2. Holding one end of the Immobiline DryStrip with forceps, carefully place the Immobiline DryStrip in-between the two glass plates of the gel. Using a thin plastic spacer, push against the plastic backing of the Immobiline DryStrip (not the gel itself) and slide the strip between the two glass plates until it comes into contact with the surface of the gel. Note: The strip should just rest on the surface of the gel. Avoid trapping air bubbles between strip and the gel and avoid piercing the second-dimension gel with the strip. 3.3 Protocol 3.3.1 Buffers Tris 25 mM; Glycine 192 mM; SDS 0.2% (w/v)pH 8.8 3.3.2 Electrophoresis conditions Running temperature: 20 °C Hold: 60 V, 1 h Hold: 200 V, 6 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 brilliant blue G-250 5.1.3 Additional reagents and buffers No additional reagents or buffer 5.1.4 Equipment No specialised equipment. 5.1.5 Direct detection protocol Temperature: 20 °C. Duration: 9 h. Detection is described in the following reference protocol: Citation: Electrophoresis,25, page(s) 327–1333 (2004). URL: not provided. 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment laser scanner 6.1.2 Name of equipment Manufacturer: Microtek scanner Model: Scan maker 9800XL Model number: 9800XL 6.1.3 Software Manufacturer: LaserSoft Imaging Model: Silverfast Ai Model number: version 6 6.1.4 Calibration No (manual) 6.1.5 Equipment specific parameters Default (vendor) parameters. 6.2 Acquisition Protocol 6.2.1 Image acquisition process Default settings with 48 bit color and 16 bit grey scale scans with atleast 300 dpi 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) Lactobacillus acidophilus NCFM reference proteome (pI 6-11) (format: TIFF) 7.1.2 Dimensions Width: 1070 px Height: 1211 px 7.1.3 Resolution 1000 ppi 7.1.4 Bit-depth 48 bits (BrilliantColor) 7.1.5 Image location Lactobacillus acidophilus NCFM reference proteome (pI 6-11).tif Link: /download/405/hBGJeKOt/ 7.1.6 Standard image orientation Yes