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. Sample name: antenna of Western Honeybee Drones (Apis mellifera ligustica)
    2. Sample type: Test sample
    1. Sample name: antenna of Western Honeybee Workers (Apis mellifera ligustica)
    2. Sample type: Test sample
    1. Sample name: antenna of Eastern honeybee Drones(Apis cerana cerana)
    2. Sample type: Test sample
    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

7.1.6 Standard image orientation

Yes

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