Name: Phakopsora pachyrhizi germinating urediniospore 2-D gel
Description: Partial proteome map by 2D gel for Phakopsora pachyrhizi germinating urediniospores.
Version: MIAPE: Gel Electrophoresis 1.4
1. General features
1.1.1 Date Stamp
2008-05-02
1.1.2 Responsible person or role
Affiliation: USDA-ARS
(i) Name: Dr. Douglas G. Luster
(ii) Postal address: USDA - ARS - FDWSRU
1301 Ditto Avenue
Ft. Detrick, MD 21702
(iii) Email address: doug.luster@ars.usda.gov
1.1.3 Electrophoresis type
Two-Dimensional electrophoresis
2. Sample
2.1.1 Sample Name(s)
-
- Sample name: Germinating spores
- Sample type: Test sample
2.1.2 Loading buffer
- DeStreak Rehydration Solution (GE Healthcare Life Sciences; Piscataway, NJ) with 1% Ampholytes
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 Life Sciences
Model: Immobiline DryStrip pH 3-10NL, 13cm
Model number: 17-6001-14
Batch number: unknown
3.2.3 Physical dimensions
X: 130 mm
Y: 3 mm
Z: 0.5 mm
3.2.4 Physiochemical property range and distribution
non-linear pH 3 - 10
3.2.5 Acrylamide concentration
4% %
3.2.6 Acrylamide : Crosslinker ratio
Crosslinker: Bisacrylamide
Ratio: 37:1
3.2.7 Additional substances in gel
No additional substance
3.2.8 Gel lane
13.2.9 Sample application
Lane 1
- Sample: Germinating spores
- Volume of sample: 400 µg
- Loading buffer: DeStreak Rehydration Solution (GE Healthcare Life Sciences; Piscataway, NJ) with 1% Ampholytes
- Volume of loading buffer: 250 µL
Loading method: rehydration loading.
Additional comment: Rehydration at 20degC, 30V for 12h
3.3 Protocol
3.3.1 Buffers
MES Running Buffer (Invitrogen; Carlsbad, CA)
3.3.2 Electrophoresis conditions
Running temperature: 20 °C
Hold: 500 V, 1 h
Hold: 1000 V, 1 h
Hold: 5000 V, 4 h
4. Inter-dimension Process
4.1 Protocol
4.1.1 Step name
reduction
4.1.2 Inter dimension buffer
50mM Tris pH 7.0, 6M Urea, 30% (w/v) glycerol, 2% SDS, 10mg/mL DTT
4.1.3 Additional reagents
No additional reagent.
4.1.4 Equipment
4.1.5 Protocol
Temperature: 25 °C.
Duration: 10 min.
Protocol:
Remove mineral oil from the IPG strips by placing them, gel side up, on a piece of dry filter paper and blotting with a second piece of dry filter paper. Place strip into test tube and add 10mL reduction buffer, rocking gently for 10 minutes on rocker.
4.1.1 Step name
alkylation
4.1.2 Inter dimension buffer
50mM Tris pH 7.0, 6M Urea, 30% (w/v) glycerol, 2% SDS, 25mg/mL iodoacetamide
4.1.3 Additional reagents
No additional reagent.
4.1.4 Equipment
4.1.5 Protocol
Temperature: 25 °C.
Duration: 10 min.
Protocol:
Remove strip from reduction buffer and place in new test tube with 10mL alkylation buffer. Rock gently for 10 minutes on rocker.
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 purchased precast.
Manufacturer: Bio-Rad Laboratories
Model: Criterion XL Precast Gel
Model number: 345-0127
Batch number: unknown
3.2.3 Physical dimensions
X: 140 mm
Y: 100 mm
Z: 1 mm
3.2.4 Physiochemical property range and distribution
linear apparent molecular mass 10 - 220 kDa
3.2.5 Acrylamide concentration
4-12% % (linear)
3.2.6 Acrylamide : Crosslinker ratio
Crosslinker: Bisacrylamide
Ratio: 37:1
3.2.7 Additional substances in gel
No additional substance
3.2.8 Gel lane
13.2.9 Sample application
Loading method: IPG strip placement.
Additional comment: Place IPG strip horizontally in IPG well; use BenchMark protein ladder (Invitrogen; Carlsbad, CA).
3.3 Protocol
3.3.1 Buffers
MES Running Buffer (Invitrogen; Carlsbad, CA)
3.3.2 Electrophoresis conditions
Running temperature: 25 °C
Hold: 120 mA, 60 min
5. Detection
5.1 Direct detection
5.1.1 Name of direct detection_process
Coomassie blue staining
5.1.2 Direct detection agents
Simply Blue Safestain (Invitrogen; Carlsbad, CA)
5.1.3 Additional reagents and buffers
Destaining solution - 4% NaCl
5.1.4 Equipment
No specialised equipment.
5.1.5 Direct detection protocol
Temperature: 25 °C.
Duration: 3 h.
Protocol:
Rinse gel 3x with 100mL deionized water. Place gel in stain for 3h. Rinse 3x. Destain in salt solution for 16h.
6. Image Acquisition
6.1 Acquisition Equipment
6.1.1 Type of equipment
camera6.1.2 Name of equipment
Manufacturer: Alpha Innotech
Model: FluorChem
Model number: 8900
6.1.3 Software
Manufacturer: Alpha Innotech
Model: FluorChem
Model number: 8900
6.1.4 Calibration
Yes (automatic)
6.1.5 Equipment specific parameters
Visual inspection6.2 Acquisition Protocol
6.2.1 Image acquisition process
Exposure time - 50msWavelength - visible light
6.2.2 Reference to gel matrix
There is only one gel in this document.
7. Image
7.1.1 Image name (or id)
050208gel (format: TIFF)
7.1.2 Dimensions
Width: 1350 px
Height: 1102 px
7.1.3 Resolution
150 ppi
7.1.4 Bit-depth
1-bit (monochrome)7.1.5 Image location
GelNoSpots091009.tif7.1.6 Standard image orientation
Yes