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Name: Analysis of leaf proteome of Vigna mungo (VM4) after MYMIV infection at 14 dpi

Description: Analysis of leaf proteome of Vigna mungo (VM4) after MYMIV infection at 14 dpi

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2009-09-10

1.1.2 Responsible person or role

Affiliation: Bose Institute

(i) Name: Prof Amita Pal

(ii) Postal address: Division of Plant Biology,
Bose Institute,
Kolkata 700054,
West Bengal, India

(iii) Email address: amita@bic.boseinst.ernet.in

1.1.3 Electrophoresis type

Two-Dimensional electrophoresis

2. Sample

2.1.1 Sample Name(s)

    1. Sample name: VM4 virus infected at 14 dpi
    2. Sample type: Test sample

2.1.2 Loading buffer

  1. 8 M Urea, 2% CHAPS, 50 mM DTT, 0.2 % Bio-lyte 3/10 ampholyte and 0.001 % 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: Bio-rad
Model: ReadyStripTM IPG Strip pH 3 - 10 Non-linear 17 cm strips
Model number: 163-2009
Batch number: Unknown

3.2.3 Physical dimensions

X: 178 mm
Y: 3.3 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: 32.3:1

3.2.7 Additional substances in gel

No additional substance

3.2.8 Gel lane

1

3.2.9 Sample application

Lane 1

  • Sample: VM4 virus infected at 14 dpi
  • Volume of sample: 400 µg
  • Loading buffer: 8 M Urea, 2% CHAPS, 50 mM DTT, 0.2 % Bio-lyte 3/10 ampholyte and 0.001 % Bromophenol Blue
  • Volume of loading buffer: 300 µL

Loading method: Passive rehydration.

3.3 Protocol

3.3.1 Buffers

No buffer.

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Gradient: 0-250 V, 15 min

Gradient: 250-10000 V, 2.5 h

Hold: 10000 V, 6 h

4. Inter-dimension Process

4.1 Protocol

4.1.1 Step name

reduction

4.1.2 Inter dimension buffer

20% v/v glycerol, 0.375 M Tris–HCl, pH 8.8, 6 M Urea, 2% (w/v) SDS, 130 mM DTT

4.1.3 Additional reagents

No additional reagent.

4.1.4 Equipment

4.1.5 Protocol

Temperature: 25 °C.

Duration: 15 min.

Protocol:
The mineral oil was removed from the IPG strips by placing them (gel side up) onto a piece of dry filter paper and blotted with a second piece of wet filter paper. 5 ml reduction buffer was added to the channeled tray. IPG strips (gel side up) were transfer into the tray. The tray was placed on an orbital shaker and gently shakes for 15 m.

4.1.1 Step name

alkylation

4.1.2 Inter dimension buffer

20% v/v glycerol, 0.375 M Tris–HCl, pH 8.8, 6 M Urea, 2% (w/v) SDS, 135 mM iodoacetamide

4.1.3 Additional reagents

No additional reagent.

4.1.4 Equipment

4.1.5 Protocol

Temperature: 25 °C.

Duration: 15 min.

Protocol:
The reduction buffer was removed carefully by decanting the liquid from the tray. 5 ml alkylation buffer was added to each IPG strip. The tray was kept on the orbital shaker for 15 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: Nature,227, page(s) 680-685 (1970).
URL: not provided.

3.2.3 Physical dimensions

X: 200 mm
Y: 180 mm
Z: 1.5 mm

3.2.4 Physiochemical property range and distribution

linear apparent molecular mass 70 - 10 kDa

3.2.5 Acrylamide concentration

1 %

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 29:1

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 transfer.

3.3 Protocol

3.3.1 Buffers

25 mM Tris, 192 mM glycine and 0.1 % SDS

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Hold: 200 V, 7.5 h

5. Detection

5.1 Direct detection

5.1.1 Name of direct detection_process

Coomassie blue staining

5.1.2 Direct detection agents

Coomassie G-250 stain

5.1.3 Additional reagents and buffers

Destaining solution: 20 % methanol, 10 % acetic acid.

5.1.4 Equipment

No specialised equipment.

5.1.5 Direct detection protocol

Temperature: 25 °C.

Duration: 12 h.

Protocol:
The gels were put into staining solution for 12 h. Destain the gels with destaining solution

6. Image Acquisition

6.1 Acquisition Equipment

6.1.1 Type of equipment

VersaDoc

6.1.2 Name of equipment

Manufacturer: Bio-rad
Model: VersaDoc Imaging System
Model number: Unknown

6.1.3 Software

Manufacturer: Bio-rad
Model: 8.0.1
Model number: Unknown

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 image was acquired on VersaDoc Imaging system using Qunatity One (Bio-rad) software with 1 s 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)

VM4 at 14 dpi (format: TIFF)

7.1.2 Dimensions

Width: 1074 px

Height: 1014 px

7.1.3 Resolution

252.2 ppi

7.1.4 Bit-depth

8-bit (256 colors)

7.1.5 Image location

VM4 14 dpi.tif

7.1.6 Standard image orientation

Yes

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