Name: Bee Hemolymph proteomics

Description: Hemolymph protein profiles of sterile and fertile honeybee workers in a queenless colony were compared by means of 2D-DIGE in order to reveal key molecular players in the repression of worker ovary activation

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2009-10-12

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. Sample name: Hemolymph of worker bees with activated ovaries
    2. Sample type: Test sample
    1. Sample name: Hemolymph of worker bees with unactivated 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: Hemolymph of worker bees with activated ovaries
  • Volume of sample: 200 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: 150 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% (w/v) 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

5.2 Indirect detection

5.2.1 Name of direct detection_process

Fluorescent detection

5.2.2 Transfer medium

N/A

Manufacturer: N/A
Model: N/A
Model number: N/A

5.2.3 Detection medium

N/A

Manufacturer: N/A
Model: N/A
Model number: N/A

5.2.4 Indirect detection agents

Deep Purple Staining (GE Healthcare)

5.2.5 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

5.2.6 Equipment

No specialised equipment.

5.2.7 Indirect 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)

HL_expasy (format: TIFF)

7.1.2 Dimensions

Width: 602 px

Height: 570 px

7.1.3 Resolution

300 ppi

7.1.4 Bit-depth

8-bit (256 colors)

7.1.5 Image location

HL_expasy.tif

7.1.6 Standard image orientation

Yes

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