Name: honeybee antenna Description: Western Honeybee Drones and Workers (Apis mellifera ligustica) Have Stronger Olfactory Functions than Their Eastern Counterparts (Apis cerana cerana): A Proteome Study Version: MIAPE: Gel Electrophoresis 1.4 1. General features ------------------- 1.1.1 Date Stamp 2010-04-15 1.1.2 Responsible person or role Affiliation: Institute of Apicultural Research (i) Name: Dr. Jianke Li (ii) Postal address: Key Laboratory of Pollinating Insect Biology, Ministry of Agriculture/Institute of Apicultural Research, Chinese Academy of Agricultural Science, Beijing 100093, China. (iii) Email address: apislijk@163.com 1.1.3 Electrophoresis type Two-Dimensional electrophoresis 2. Sample --------- 2.1.1 Sample Name(s) 1. 1. Sample name: antenna of Western Honeybee Drones (Apis mellifera ligustica) 2. Sample type: Test sample 2. 1. Sample name: antenna of Western Honeybee Workers (Apis mellifera ligustica) 2. Sample type: Test sample 3. 1. Sample name: antenna of Eastern honeybee Drones(Apis cerana cerana) 2. Sample type: Test sample 4. 1. Sample name: antenna of Eastern honeybee workers (Apis cerana cerana) 2. Sample type: Test sample 2.1.2 Loading buffer 1. 8 M urea, 2% CHAPS, 0.001% bromophenol blue, 45 mM DTT, 0.2% Bio-lyte pH 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: Bio-Rad Model: Bio-Rad Model number: ReadyStrip IPG Strips 17cm, pH 3-10 12 pack Batch number: UNKNOWN 3.2.3 Physical dimensions X: 178 mm Y: 33 mm Z: 0.5 mm 3.2.4 Physiochemical property range and distribution logarithmic apparent molecular mass 100 - 10 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 1.5mTris8.8,10%SDS,TEMED,10%AP 3.2.8 Gel lane 1 3.2.9 Sample application Lane 1 * Sample: antenna of Western Honeybee Drones (Apis mellifera ligustica) * Volume of sample: 550 µg * Loading buffer: 8 M urea, 2% CHAPS, 0.001% bromophenol blue, 45 mM DTT, 0.2% Bio-lyte pH 3-10 * Volume of loading buffer: 336 µL Loading method: rehydration loading. 3.3 Protocol 3.3.1 Buffers 8M urea,2M thiourea, 4%CHAPS, 20mM Trisbase, 30mM DTT, 0.5%Bio-lyte(pH 3.5-10) 3.3.2 Electrophoresis conditions Running temperature: 18 °C Hold: 50 V, 14 h 4. Inter-dimension Process -------------------------- 4.1 Protocol 4.1.1 Step name reduction and alkylation 4.1.2 Inter dimension buffer equilibration buffer 1 (reduction buffer):6 M urea, 0.375 M Tris-HCl pH 8.8, 20% glycerol, 2% SDS, 2% DTT equilibration buffer 2 (alkylation buffer):(6 M urea, 0.375 M Tris-HCl pH 8.8, 20% glycerol, 2% SDS, 2.5% iodoacetoamide 4.1.3 Additional reagents equilibration buffer 1 (reductionbuffer):6 M urea, 0.375 M Tris-HCl pH 8.8, 20% glycerol, 2% SDS, 2% DTT equilibration buffer 2 (alkylation buffer):(6 M urea, 0.375 M Tris-HCl pH 8.8, 20% glycerol, 2% SDS, 2.5% iodoacetoamide 4.1.4 Equipment Manufacturer: 13 Model: shaker Model number: 12 4.1.5 Protocol Temperature: 25 °C. Duration: 15 min. Protocol: 15 minutes in equilibration buffer each 4.1.1 Step name reduction and alkylation 4.1.2 Inter dimension buffer equilibration buffer 1 (reduction buffer):6 M urea, 0.375 M Tris-HCl pH 8.8, 20% glycerol, 2% SDS, 2% DTT equilibration buffer 2 (alkylation buffer):(6 M urea, 0.375 M Tris-HCl pH 8.8, 20% glycerol, 2% SDS, 2.5% iodoacetoamide 4.1.3 Additional reagents No additional reagent. 4.1.4 Equipment Manufacturer: 13 Model: shaker Model number: 12 4.1.5 Protocol Temperature: 25 °C. Duration: 15 min. Protocol: 15 minutes in equilibration buffer each 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 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: Bio-Rad Model: Bio-Rad Model number: ReadyStrip IPG Strips 17cm, pH 3-10 12 pack Batch number: unknown 3.2.3 Physical dimensions X: 178 mm Y: 33 mm Z: 0.5 mm 3.2.4 Physiochemical property range and distribution logarithmic apparent molecular mass 100 - 10 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 1.5mTris8.8,10%SDS,TEMED,10%AP 3.2.8 Gel lane 1 3.2.9 Sample application Loading method: rehydration loading. 3.3 Protocol 3.3.1 Buffers 8M urea,2M thiourea, 4%CHAPS, 20mM Trisbase, 30mM DTT, 0.5%Bio-lyte(pH 3.5-10) 3.3.2 Electrophoresis conditions Running temperature: 18 °C Hold: 50 V, 14 h Hold: 250 V, 2 h Hold: 1000 V, 1 h Hold: 9000 V, 5 h Hold: 9000 V, 6.5 h 5. Detection ------------ 5.1 Direct detection 5.1.1 Name of direct detection_process Fluorescent staining 5.1.2 Direct detection agents Flamingo fluorescent dye 5.1.3 Additional reagents and buffers No additional reagents or buffer 5.1.4 Equipment Manufacturer: Bio-Rad Model: pharos Fx plus Model number: unknown Manufacturer: GE Healthcare Model: Image Scanner III Model number: unknown 5.1.5 Direct detection protocol Temperature: 25 °C. Duration: 10 min. Protocol: Flamingo:water=1:10 for 30 min 6. Image Acquisition -------------------- 6.1 Acquisition Equipment 6.1.1 Type of equipment laser scanner 6.1.2 Name of equipment Manufacturer: Bio-Rad Model: pharos Fx plus Model number: unknown 6.1.3 Software Manufacturer: Bio-Rad Model: quantity one Model number: 4.6.6 6.1.4 Calibration Yes (automatic) 6.1.5 Equipment specific parameters 532 nm EX 100 micrometer 6.2 Acquisition Protocol 6.2.1 Image acquisition process 532 nm EX 100 micrometer 6.2.2 Reference to gel matrix There is only one gel in this document. 7. Image -------- 7.1.1 Image name (or id) antenna (format: JPEG) 7.1.2 Dimensions Width: 2450 px Height: 2550 px 7.1.3 Resolution 100 ppi 7.1.4 Bit-depth 8-bit (256 colors) 7.1.5 Image location C:\\\\Documents and Settings\\\\Administrator\\\\桌面\\\\male 091016-4.jpg Link: /download/520/980SaSTG/ 7.1.6 Standard image orientation Yes