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Name: Analysis of freezing stress modulated secretome of Hippophae rhamnoides

Description: Twenty days old seabuckthorn seedlings were given -5 °C treatment and extracellular proteins were extracted. 250 µg of the proteins were loaded on 3-10 nonlinear IPG strip. Second dimension was carried out at 15% SDS-PAGE and the gel was silver stained.

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2010-04-01

1.1.2 Responsible person or role

Affiliation: University of Delhi

(i) Name: Dr. Renu Deswal

(ii) Postal address: Molecular Plant Physiology and Proteomics Laboratory, Department of Botany, University of Delhi

(iii) Email address: rdeswal@botany.du.ac.in

1.1.3 Electrophoresis type

Two-Dimensional electrophoresis

2. Sample

2.1.1 Sample Name(s)

    1. Sample name: Freezing stress modulated extracellular (apoplastic) protein from shoot of Hippophae rhamnoides seedlings
    2. Sample type: Test sample
    3. URL: http://pubs.acs.org/doi/abs/10.1021/pr200944z?mi=qryllt&af=R&pageSize=20&searchText=ATP

2.1.2 Loading buffer

  1. 8 M urea, 2 M thiourea, 2% w/v CHAPS, 50 mM DTT, 0.5% v/v IPG buffer, 2% Triton X-100 and a trace of bromophenol blue

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: G E Healthcare Life Sciences
Model: IPG Strip 3-10 non linear
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: 32:1

3.2.7 Additional substances in gel

No additional substance

3.2.8 Gel lane

1

3.2.9 Sample application

Lane 1

  • Sample: Freezing stress modulated extracellular (apoplastic) protein from shoot of Hippophae rhamnoides seedlings
  • Volume of sample: 250 µg
  • Loading buffer: 8 M urea, 2 M thiourea, 2% w/v CHAPS, 50 mM DTT, 0.5% v/v IPG buffer, 2% Triton X-100 and a trace of bromophenol blue
  • Volume of loading buffer: 250 µL

Loading method: rehydration loading.

3.3 Protocol

3.3.1 Buffers

No buffer.

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Hold: 500 V, 4 h

Gradient: 500-1000 V, 1.30 h

Gradient: 1000-8000 V, 2.3 h

Hold: 8000 V, 0.3 h

4. Inter-dimension Process

4.1 Protocol

4.1.1 Step name

equilibration

4.1.2 Inter dimension buffer

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

4.1.3 Additional reagents

Reduction-10 mg/mL DTT
Alkylation-25 mg/mL iodoacetamide

4.1.4 Equipment

Manufacturer: No Specialised equipment was used
Model: No Specialised equipment was used
Model number: No Specialised equipment was used

4.1.5 Protocol

Temperature: 20 °C.

Duration: 20 min.

Protocol:
Strip was first equilibrated with 5 mL equilibration buffer A containing 10 mg/mL DTT for 20 min and then replaced with equilibration buffer B containing 25 mg/mL iodoacetamide for next 20 min.

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 reference protocol:

Citation: CSH Protocols,doi:10.1101pdb.prot4540, page(s) doi:10.1101pdb.prot4540 (2006).
URL: http://vetbiotech.um.ac.ir/parameters/vetbiotech/filemanager/new_admin/electrophorsis/SDS-Polyacrylamide%20Gel%20Electrophoresis%20of%20Proteins.pdf

3.2.3 Physical dimensions

X: 180 mm
Y: 160 mm
Z: 1 mm

3.2.4 Physiochemical property range and distribution

logarithmic apparent molecular mass 14.2 - 97 kDa

3.2.5 Acrylamide concentration

15 %

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 30:0.8

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 transfer.

3.3 Protocol

3.3.1 Buffers

25 mM Tris
192 mM Glycine
0.1% SDS

3.3.2 Electrophoresis conditions

Running temperature: room temperature (no cooling device).

Hold: 10 mA, 0.3 h

Hold: 20 mA, 6 h

5. Detection

5.1 Direct detection

5.1.1 Name of direct detection_process

Silver staining

5.1.2 Direct detection agents

Silver nitrate

5.1.3 Additional reagents and buffers

45% methnol and 10% acetic acid (fixing solution), 20 mg/100 mL sodium thiosulphate (sensitizer), 2 g/100 mL (developer) and 10% acetic acid (Stopper)

5.1.4 Equipment

Manufacturer: No Specialised equipment was used
Model: No Specialised equipment was used
Model number: No Specialised equipment was used

5.1.5 Direct detection protocol

Temperature: 4 °C.

Duration: 10 min.

Detection is described in the following reference protocol:
Citation: Methods in enzymology,96, page(s) 230-239 (1983).
URL: not provided.

6. Image Acquisition

6.1 Acquisition Equipment

6.1.1 Type of equipment

camera

6.1.2 Name of equipment

Manufacturer: AlphaImager gel documentation system
Model: Alpha imager
Model number: AlphaImager HP System

6.1.3 Software

Manufacturer: AlphaImager gel documentation system
Model: Alpha View Software
Model number: Alpha View Software

6.1.4 Calibration

Yes (automatic)

6.1.5 Equipment specific parameters

Default (vendor) parameters.

Configuration file: Freezing stress modulated extracellular proteins of Hippophae rhamnoides

6.2 Acquisition Protocol

6.2.1 Image acquisition process

Image was acquired 2 sec exposure time

6.2.2 Reference to gel matrix

There is only one gel in this document.

7. Image

7.1.1 Image name (or id)

HRHAMNOIDES_SEEDLINGS (ECP)_3-10 (format: TIFF)

7.1.2 Dimensions

Width: 1153 px

Height: 1000 px

7.1.3 Resolution

102 ppi

7.1.4 Bit-depth

8-bit (256 colors)

7.1.5 Image location

http://pubs.acs.org/doi/abs/10.1021/pr200944z?mi=qryllt&af=R&pageSize=20&searchText=ATP

7.1.6 Standard image orientation

Yes

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