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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)

    1. Sample name: Germinating spores
    2. Sample type: Test sample

2.1.2 Loading buffer

  1. 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

1

3.2.9 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

1

3.2.9 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

camera

6.1.2 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 inspection

6.2 Acquisition Protocol

6.2.1 Image acquisition process

Exposure time - 50ms
Wavelength - 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.tif

7.1.6 Standard image orientation

Yes

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