Name: Ovary workerbee proteome Description: Proteome of the ovaries developed and rudimentary of workerbees Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2008-08-08 1.1.2 Responsible person or role Affiliation: Katholieke Universiteit Leuven (i) Name: Prof. Dr. Liliane Schoofs (ii) Postal address: Department for Biology Research group of Functional Genomics and Proteomics Naamsestraat 59 3000 Leuven Belgium (iii) Email address: liliane.schoofs@bio.kuleuven.be 1.1.3 Electrophoresis type Two-Dimensional electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: proteome of developed and rudimentary honeybee worker ovaries 2. Sample type: Control sample 2.1.2 Loading buffer 1. Urea (7 M) Thiourea (2 M) 4% CHAPS 40 nM Tris 1 % DTT Complete Protease Inhibitor Cocktail, Roche Diagnostics GmbH 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 Bio-Sciences Corp. Model: ImmobilineTM Drystrip pH 3-10 Non-linear 24 cm strips Model number: 17-6002-45 Batch number: unknown 3.2.3 Physical dimensions X: 240 mm Y: 3 mm Z: 0.5 mm 3.2.4 Physiochemical property range and distribution sigmoidal 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 Destreak rehydration solution (GE Healthcare, 17-6003-19) IPG Buffer pH 3-10 NL (GE Healthcare, 17-6000-88) 3.2.8 Gel lane 1 3.2.9 Sample application Lane 1 * Sample: proteome of developed and rudimentary honeybee worker ovaries * Volume of sample: 500 µg * Loading buffer: Urea (7 M) Thiourea (2 M) 4% CHAPS 40 nM Tris 1 % DTT Complete Protease Inhibitor Cocktail, Roche Diagnostics GmbH * Volume of loading buffer: 180 µL Loading method: cup loading. 3.3 Protocol 3.3.1 Buffers No buffer. 3.3.2 Electrophoresis conditions Running temperature: 20 °C Hold: 150 V, 3 h Hold: 300 V, 3 h Hold: 1000 V, 6 h Hold: 8000 V, 6 h 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name equilibration 4.1.2 Inter dimension buffer Buffer A: 6 M urea, 30% (v/v) glycerol, 2% SDS, 50 mM Tris-HCl (pH 8.8), 1% (w/v) DTT Buffer B: buffer A in which DTT is replaced by 4% (w/v) iodoacetamide 4.1.3 Additional reagents Bromophenol blue added in the second part of the equilibration step, as a tracking dye for the second dimension separation. 4.1.4 Equipment Manufacturer: GE Healthcare Bio-Sciences Corp. Model: Immobiline™ DryStrip Reswelling Tray Model number: 80-6465-32 4.1.5 Protocol Temperature: 20 °C. Duration: 30 min. Protocol: 15 minutes incubation in buffer A, followed by 15 minutes incubation in buffer B (whith added bromophenol blue) 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: 30% acrylamide/bisacrylamide (250 ml); Tris-Cl pH 8.8 (150 ml); MilliQ (millipore) (187 ml); 10% sds (6 ml); 10% ammonium persulfate (6 ml): 10% TEMED (830 µl) 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 10 - 200 kDa 3.2.5 Acrylamide concentration 12.50% % 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 transfer. 3.3 Protocol 3.3.1 Buffers 3x SDS 1x SDS 3.3.2 Electrophoresis conditions Running temperature: 20 °C Hold: 8 mA, 1 h Hold: 12 mA, 15.00 h 5. Detection ------------ 5.1 Direct detection 5.1.1 Name of direct detection_process Fluorescent staining 5.1.2 Direct detection agents Deep Purple Staining (GE Healthcare) 5.1.3 Additional reagents and buffers A: 10% methanol / 7.5% acetic acid (v/v) in Milli-Q (Millipore) B: 0.035 M NaHCO3 + 0.30 M Na2CO3 in Milli-Q C: 1/200 dilution of Deep Purple in Milli-Q D: 7.5% acetic acid in Milli-Q 5.1.4 Equipment No specialised equipment. 5.1.5 Direct detection protocol Temperature: 20 °C. Duration: 3 h. Protocol: The complete protocol is carried out at room temperature and gels are incubated in the different solutions while shaking gently fixation of the gels: minimum 1 h in solution A (make sure the gel is submersed in the solution) at room temperature wash: remove solution A and wash in solution B for 30 minutes at room temperature staining: 1 h. in solution C, keep gels in a dark place to prevent bleaching fading: remove solution C and add solutioni D, 15 minutes incubation in the dark. Repeat this once. Store gel in solution D at 4�C 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment fluorescent scanner 6.1.2 Name of equipment Manufacturer: GE Healthcare Model: Ettan™ DIGE Imager Model number: 63-0056-42 6.1.3 Software Manufacturer: GE Healthcare Model: Decyder 2D 7.0 Model number: 28-9435-86 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 excitation: 532 nm emission: 560 LP or 610 BP filter exposure time: 0.800 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) 1000x1000 (format: TIFF) 7.1.2 Dimensions Width: 999 px Height: 999 px 7.1.3 Resolution 100 µm/px 7.1.4 Bit-depth 8-bit (256 colors) 7.1.5 Image location 1000x1000.tif Link: /download/304/pw39CJRN/ 7.1.6 Standard image orientation Yes