Name: Two-dimensional gel of Spartina alterniflora

Description: Heat-stable proteins (95C for 40 min) were extracted from Spartina alterniflora, and separated on two-dimensinoal gels (pI 3-10 NL, 24 cm IPG).

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2011-01-05

1.1.2 Responsible person or role

Affiliation: Louisiana State University, AgCenter

(i) Name: Wang, Yi

(ii) Postal address: Department of Plant Pathology and Crop Physiology
302 Life Sciences Building
Louisiana State University
Baton Rouge, LA 70803

(iii) Email address: ywang17@tigers.lsu.edu

1.1.3 Electrophoresis type

Two-Dimensional electrophoresis

2. Sample

2.1.1 Sample Name(s)

    1. Sample name: Heat stable proteins from Spartina alterniflora seeds
    2. Sample type: Test sample

2.1.2 Loading buffer

  1. 7M urea, 2M thiourea, 4% CHAPS (w/v) and 20mM dithiothreitol (DTT), 2% (w/v) IPG buffer NL 3-10. Rehydration buffer: 7 M urea, 2 M thiourea, 0.001% (w/v) bromophenol, 2% (w/v) CHAPS, 20 mM DTT and 0.5% (w/v) IPG buffer NL 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: GE Healthcare
Model: Immobiline DryStrip pH 3-10 NL, 24 cm
Model number: 17-6002-45
Batch number: 10057246

3.2.3 Physical dimensions

X: 240 mm
Y: 3 mm
Z: 0.5 mm

3.2.4 Physiochemical property range and distribution

NL 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

No additional substance

3.2.8 Gel lane

5

3.2.9 Sample application

Lane 1

  • Sample: Heat stable proteins from Spartina alterniflora seeds
  • Volume of sample: 1000 µg
  • Loading buffer: 7M urea, 2M thiourea, 4% CHAPS (w/v) and 20mM dithiothreitol (DTT), 2% (w/v) IPG buffer NL 3-10. Rehydration buffer: 7 M urea, 2 M thiourea, 0.001% (w/v) bromophenol, 2% (w/v) CHAPS, 20 mM DTT and 0.5% (w/v) IPG buffer NL 3-10.
  • Volume of loading buffer: 30 µL

Lane 2

  • Sample: Heat stable proteins from Spartina alterniflora seeds
  • Volume of sample: 1000 µg
  • Loading buffer: 7M urea, 2M thiourea, 4% CHAPS (w/v) and 20mM dithiothreitol (DTT), 2% (w/v) IPG buffer NL 3-10. Rehydration buffer: 7 M urea, 2 M thiourea, 0.001% (w/v) bromophenol, 2% (w/v) CHAPS, 20 mM DTT and 0.5% (w/v) IPG buffer NL 3-10.
  • Volume of loading buffer: 30 µL

Lane 3

  • Sample: Heat stable proteins from Spartina alterniflora seeds
  • Volume of sample: 1000 µg
  • Loading buffer: 7M urea, 2M thiourea, 4% CHAPS (w/v) and 20mM dithiothreitol (DTT), 2% (w/v) IPG buffer NL 3-10. Rehydration buffer: 7 M urea, 2 M thiourea, 0.001% (w/v) bromophenol, 2% (w/v) CHAPS, 20 mM DTT and 0.5% (w/v) IPG buffer NL 3-10.
  • Volume of loading buffer: 30 µL

Lane 4

  • Sample: Heat stable proteins from Spartina alterniflora seeds
  • Volume of sample: 1000 µg
  • Loading buffer: 7M urea, 2M thiourea, 4% CHAPS (w/v) and 20mM dithiothreitol (DTT), 2% (w/v) IPG buffer NL 3-10. Rehydration buffer: 7 M urea, 2 M thiourea, 0.001% (w/v) bromophenol, 2% (w/v) CHAPS, 20 mM DTT and 0.5% (w/v) IPG buffer NL 3-10.
  • Volume of loading buffer: 30 µL

Lane 5

  • Sample: Heat stable proteins from Spartina alterniflora seeds
  • Volume of sample: 1000 µg
  • Loading buffer: 7M urea, 2M thiourea, 4% CHAPS (w/v) and 20mM dithiothreitol (DTT), 2% (w/v) IPG buffer NL 3-10. Rehydration buffer: 7 M urea, 2 M thiourea, 0.001% (w/v) bromophenol, 2% (w/v) CHAPS, 20 mM DTT and 0.5% (w/v) IPG buffer NL 3-10.
  • Volume of loading buffer: 30 µL

Loading method: rehydration loading.
Additional comment: Immobiline DryStrip IPGbox rehydration for 12 hours.

3.3 Protocol

3.3.1 Buffers

No buffer.

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Hold: 500 V, 2 h

Hold: 1000 V, 2 h

Gradient: 1000-8000 V, 2 h

Hold: 8000 V, 3 h

Gradient: 8000-10000 V, 1 h

Hold: 10000 V, 1 h

4. Inter-dimension Process

4.1 Protocol

4.1.1 Step name

reduction

4.1.2 Inter dimension buffer

50 mM Tris-HCl (pH 8.8), 6 M urea, 30% glycerol (v/v), 2% SDS (w/v), 0.002% (w/v) bromophenol

4.1.3 Additional reagents

0.5% DTT (w/v)

4.1.4 Equipment

Manufacturer: unknown
Model: Equilibrium tray 12 wells
Model number: unknown

4.1.5 Protocol

Temperature: 22 °C.

Duration: 30 min.

Protocol:
Strips immersed in individual wells of a rehydration tray and kept shaking for 30 minutes.

4.1.1 Step name

alkylation

4.1.2 Inter dimension buffer

50 mM Tris-HCl (pH 8.8), 6 M urea, 30% glycerol (v/v), 2% SDS (w/v), 0.002% (w/v) bromophenol

4.1.3 Additional reagents

1.25% Iodoacetamide (w/v)

4.1.4 Equipment

Manufacturer: unknown
Model: Equilibrium tray 12 wells
Model number: unknown

4.1.5 Protocol

Temperature: 22 °C.

Duration: 40 min.

Protocol:
Strips immersed in individual wells of a rehydration tray and kept shaking for 40 minutes.

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:

A recipe to prepare 1200 ml of 12.5% polyacrylamide gels [150.0 g acrylamide, 4.032 g bis-acrylamide, 1.5 M Tris-HCl pH 8.8, 10% SDS (w/v), 10% APS (w/v) and 340 µl TEMED] (the stirred gel solution was degassed under vacuum for 20 minutes, and APS and TEMED were not added until gels were ready to pour) was used to make 12 gels.

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 100 - 6 kDa

3.2.5 Acrylamide concentration

12.5 %

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 37.2:1

3.2.7 Additional substances in gel

No additional substance

3.2.8 Gel lane

5

3.2.9 Sample application

Loading method: IPG strip.
Additional comment: IPG strip was placed on top of the slab gel and held in place with Agarose Sealing Solution (0.5% agarose in 1x SDS Electrophoresis Buffer).

3.3 Protocol

3.3.1 Buffers

1x SDS running buffer: 2.5 mM Tris-base pH 8.3, 19.2 mM glycine and 0.01 % (w/v) SDS.
2x SDS topping buffer: 5 mM Tris-base pH 8, 38.4 mM glycine and 0.02% (w/v) SDS.

3.3.2 Electrophoresis conditions

Running temperature: 18 °C

Hold: 40 V, 1 h

Hold: 110 V, 13 h

5. Detection

5.1 Direct detection

5.1.1 Name of direct detection_process

Colloidal Coomassie Blue G-250 staining (Blue silver staining)

5.1.2 Direct detection agents

2% H3PO4, 10% (NH4)2SO4, 20% methanol and 0.1% Coomassie G-250.

The preparation of the staining recipe (10 L) was by sequentially addition as follows: 235 ml of phosphoric acid (85%, w/v) was added to 2 L of distilled water; 1 kg of ammonium sulfate powder was added to 4 L of distilled water. After they were fully dissolved, phosphoric acid and ammonium sulfate solutions were combined and poured into the staining tank together with 1.5 L of distilled water. Ten grams of Coomassie Brilliant Blue G-250 were dissolved in 500 ml distilled water, and then added to staining tank. Two liters of methanol were not added to staining tank until the gel staining began.

5.1.3 Additional reagents and buffers

No additional reagents or buffer

5.1.4 Equipment

Manufacturer: Bio-Rad
Model: Dodeca Stainer
Model number: #165-3401

5.1.5 Direct detection protocol

Temperature: 22 °C.

Duration: 4 d.

Protocol:
The staining took place for 2-3 days, and gels were de-stained with distilled water until the background was clear (8-10 hours in most cases).

6. Image Acquisition

6.1 Acquisition Equipment

6.1.1 Type of equipment

Professional scanner

6.1.2 Name of equipment

Manufacturer: UMAX
Model: UTA
Model number: 1100

6.1.3 Software

Manufacturer: UMAX
Model: Magicscan
Model number: 4.5

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

The gel images were acquired in 16 bit grayscale with a resolution of 300 dpi and were saved in tiff format. No filters or image processing tools were used during acquisition.

6.2.2 Reference to gel matrix

There is only one gel in this document.

7. Image

7.1.1 Image name (or id)

SA Gel #1 - #5 (format: TIFF)

7.1.2 Dimensions

Width: 2196 px

Height: 2954 px

7.1.3 Resolution

300 dpi

7.1.4 Bit-depth

16-bit (HighColor)

7.1.5 Image location

sa #1.tif

7.1.6 Standard image orientation

Yes

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