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)
-
- Sample name: Hemolymph of worker bees with activated ovaries
- Sample type: Test sample
-
- Sample name: Hemolymph of worker bees with unactivated ovaries
- Sample type: Control sample
2.1.2 Loading buffer
- 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
13.2.9 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
13.2.9 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 scanner6.1.2 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 nmemission: 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.tif7.1.6 Standard image orientation
Yes