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)
-
- Sample name: Freezing stress modulated extracellular (apoplastic) protein from shoot of Hippophae rhamnoides seedlings
- Sample type: Test sample
- URL: http://pubs.acs.org/doi/abs/10.1021/pr200944z?mi=qryllt&af=R&pageSize=20&searchText=ATP
2.1.2 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
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
13.2.9 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
13.2.9 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
camera6.1.2 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 time6.2.2 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=ATP7.1.6 Standard image orientation
Yes