Name: Basic proteins of the dlPFC region of the human brain Description: Basic proteins (pH 6-11) of the dorso lateral prefrontal cortex region of the human brain separated by 2-DE Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2008-04-01 1.1.2 Responsible person or role Affiliation: UCD (i) Name: Ciara McManus (ii) Postal address: UCD Conway Institute of Biomolecular and Biomedical Research, UCD, Belfield, Dublin, Ireland (iii) Email address: ciara.mcmanus@ucd.ie 1.1.3 Electrophoresis type Two-Dimensional electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: Dorso later prefrontal cortex (dlPFC) region of the human brain 2. Sample type: Control sample 3. URL: http://www.stanleyresearch.org/dnn/ Link: http://www.stanleyresearch.org/dnn/ 2.1.2 Loading buffer 1. 9.5 M urea, 2% CHAPS, 0.8% Pharmalyte pH 3-10, 1% DTT, Protease Inhibitors 2. 8 M urea, 0.5% w/v CHAPS, 0.2% w/v DTT, 0.2% w/v 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 Healthcare Model: Immobiline DryStrip pH 6-9, 18 cm Model number: 17-6001-97 Batch number: 10008430 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: 1:3 3.2.7 Additional substances in gel DeStreak rehydration solution (GE Healthcare, 17-6003-19) 3.2.8 Gel lane 1 3.2.9 Sample application Lane 1 * Sample: Dorso later prefrontal cortex (dlPFC) region of the human brain * Volume of sample: 300 µg * Loading buffer: 8 M urea, 0.5% w/v CHAPS, 0.2% w/v DTT, 0.2% w/v Pharmalyte pH 6-11 * Volume of loading buffer: 350 µL Loading method: paper bridge loading. 3.3 Protocol 3.3.1 Buffers No buffer. 3.3.2 Electrophoresis conditions Running temperature: 20 °C Hold: 200 V, 16 h Hold: 150 V, 1 h Hold: 300 V, 1 h Hold: 600 V, 1 h Hold: 3500 V, 75 h Hold: 8000 V, 10 min Hold: 8000 V, 3 h Hold: 100 V, 72 h 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name reduction 4.1.2 Inter dimension buffer 6 M urea containing 30% v/v glycerol, 2% w/v SDS, 0.05 M Tris-HCl, pH 8.8 and 0.01% w/v Bromophenol blue with the addition of 1% w/v DTT 4.1.3 Additional reagents No additional reagent. 4.1.4 Equipment 4.1.5 Protocol Temperature: 22 °C. Duration: 15 min. 4.1.1 Step name alkylation 4.1.2 Inter dimension buffer 6 M urea containing 30% v/v glycerol, 2% w/v SDS, 0.05 M Tris-HCl, pH 8.8 and 0.01% w/v Bromophenol blue with the addition of 4.7% w/v iodoacetamide 4.1.3 Additional reagents No additional reagent. 4.1.4 Equipment 4.1.5 Protocol Temperature: 22 °C. Duration: 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: http://nationaldiagnostics.com/images/ec890_protocol.pdf,http://nationaldiagnostics.com/images/ec890_protocol.pdf, page(s) http://nationaldiagnostics.com/images/ec890_protocol.pdf (2005). URL: http://nationaldiagnostics.com/images/ec890_protocol.pdf Link: http://nationaldiagnostics.com/images/ec890_protocol.pdf 3.2.3 Physical dimensions X: 240 mm Y: 200 mm Z: 1 mm 3.2.4 Physiochemical property range and distribution logarithmic apparent molecular mass 6 - 150 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: paper bridge loading. 3.3 Protocol 3.3.1 Buffers 25mM Tris, 192mM Glycine and 0.1% SDS. 3.3.2 Electrophoresis conditions Running temperature: 15 °C Hold: 150 mA, 16 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 Fixer 40% Ethanol, 10% Acetic Acid, ddH2O Sensitising Sodium Acetate, Sodium Thiosulphate, Ethanol Milli-Q Developing Sodium Carbonate, formaldehyde Stopping - EDTA 5.1.4 Equipment No specialised equipment. 5.1.5 Direct detection protocol Temperature: 22 °C. Duration: 5 h. Detection is described in the following reference protocol: Citation: Electrophoresis,21, page(s) 3666-3672 (2000). URL: http://www3.interscience.wiley.com/journal/75505007/abstract Link: http://www3.interscience.wiley.com/journal/75505007/abstract 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment Calibrated densitometer 6.1.2 Name of equipment Manufacturer: Bio-Rad Model: GS-800 Calibrated Densitometer Model number: 170-7980 6.1.3 Software Manufacturer: Bio-Rad Model: PDQuest Model number: 7.3.1 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 Filter = Green, Light = Transmissive 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) Brain dlPFC 6-11 (format: PNG) 7.1.2 Dimensions Width: 550 px Height: 568 px 7.1.3 Resolution 72 dpi 7.1.4 Bit-depth 8-bit (256 colors) 7.1.5 Image location C:\Documents and Settings\Ciara McManus\My Documents\Bridge loading 6-11 gels (stanley series)\Database\BRAIN_DLPFC_6-11.png Link: /download/247/i8W6rpse/ 7.1.6 Standard image orientation Yes