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Name: TCA-acetone_3

Description: TCA-acetone extraction of Arabidopsis leaf proteome

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2007-04-30

1.1.2 Responsible person or role

Affiliation: Agricultural and Plant Biochemistry and Proteomics Research Group, Dept. Biochemistry and Molecular Biology, University of Córdoba, Spain

(i) Name: Prof. Jesus V. Jorrin Novo

(ii) Postal address: Dpto. Bioquimica y Biologia Molecular, Universidad de Cordoba, Campus de Rabanales, edificio Severo Ochoa, planta baja, 14014, Cordoba, Spain

(iii) Email address: bf1jonoj@uco.es

1.1.3 Electrophoresis type

Two-Dimensional electrophoresis

2. Sample

2.1.1 Sample Name(s)

    1. Sample name: Arabidopsis thaliana leaf proteins extracted with a TCA-acetone precipitation method
    2. Sample type: Test sample

2.1.2 Loading buffer

  1. Urea 9 M, CHAPS 4 %, DTT 20 mM, Ampholytes 2 %, bromophenol blue 0.005 %

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: BioRad
Model: IPG strip 17 cm pH 5-8
Model number: 163-2011
Batch number: unknown

3.2.3 Physical dimensions

X: 17.1 cm
Y: 0.33 cm
Z: 0.05 cm

3.2.4 Physiochemical property range and distribution

linear pH 5 - 8

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: Arabidopsis thaliana leaf proteins extracted with a TCA-acetone precipitation method
  • Volume of sample: 100 µg
  • Loading buffer: Urea 9 M, CHAPS 4 %, DTT 20 mM, Ampholytes 2 %, bromophenol blue 0.005 %
  • Volume of loading buffer: 300 µL

Loading method: rehydration loading.

3.3 Protocol

3.3.1 Buffers

No buffer.

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Gradient: 0-250 V, 20 min

Gradient: 250-10000 V, 2.5 h

Hold: 10000 V, 3.5 h

4. Inter-dimension Process

4.1 Protocol

4.1.1 Step name

reduction

4.1.2 Inter dimension buffer

Tris-HCl 375 mM pH 8.8, urea 6 M, SDS 2%, glycerol 20 %, DTT 2%

4.1.3 Additional reagents

No additional reagent.

4.1.4 Equipment

4.1.5 Protocol

Temperature: 25 °C.

Duration: 15 min.

Protocol:
Remove the mineral oil from the IPG strips by placing them (gel side up) onto a piece of dry filter paper
and blotting with a second piece of wet filter paper. Add 4 ml reduction buffer to laned tray, using one channel per IPG strip. Transfer the blotted IPG strips (gel side up)
into the tray. Place the tray on an orbital shaker and gently shake for 15 m.

4.1.1 Step name

alkylation

4.1.2 Inter dimension buffer

Tris-HCl 375 mM pH 8.8, urea 6 M, SDS 2%, glycerol 20 %, DTT 2,5 %

4.1.3 Additional reagents

No additional reagent.

4.1.4 Equipment

4.1.5 Protocol

Temperature: 25 °C.

Duration: 15 min.

Protocol:
Discard the used reduction buffer by carefully decanting the
liquid from the tray. Remove the last few drops of reduction buffer. Add the indicated volume alkylation buffer to each IPG
strip. Return the tray to 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 receipe:

34 % of destilated water
40 % of a 30 % acrylamide/Bis solution
25 % of 1.5 M Tris-HCl pH 8.8
1 % of a 10 % SDS solution
0.5 % of a 10% APS solution
0.05% of TEMED

3.2.3 Physical dimensions

X: 20 cm
Y: 20 cm
Z: 0.1 cm

3.2.4 Physiochemical property range and distribution

linear apparent molecular mass 100 - 12 kDa

3.2.5 Acrylamide concentration

12 %

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 37.5:1

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 placing.

3.3 Protocol

3.3.1 Buffers

0.025 M Tris-HCl pH 8.3, 0.192 M glycine, 0.1 % SDS

3.3.2 Electrophoresis conditions

Running temperature: 18 °C

Hold: 600 mA, 18 h

5. Detection

5.1 Direct detection

5.1.1 Name of direct detection_process

Fluorescent staining

5.1.2 Direct detection agents

SyPro Ruby, Sigma

5.1.3 Additional reagents and buffers

Fixation and destaining solution: 10 % methanol, 7 % acetic acid.

5.1.4 Equipment

No specialised equipment.

5.1.5 Direct detection protocol

Temperature: 25 °C.

Duration: 18 h.

Protocol:
Put the gels in fixing solution during 30 min. Stain the gels in SyPro Ruby solution during 18 h, kept from light. Destain the gels with destaining solution during 30 min, kept from light.

6. Image Acquisition

6.1 Acquisition Equipment

6.1.1 Type of equipment

fluorescent scanner

6.1.2 Name of equipment

Manufacturer: BioRad
Model: Molecular Imager FX Pro Plus multiImager System
Model number: unknown

6.1.3 Software

Manufacturer: BioRad
Model: PDQuest
Model number: 7.1

6.1.4 Calibration

Yes (automatic)

6.1.5 Equipment specific parameters

Visual inspection of the matching process

6.2 Acquisition Protocol

6.2.1 Image acquisition process

100x100 microns

6.2.2 Reference to gel matrix

There is only one gel in this document.

7. Image

7.1.1 Image name (or id)

TCA-acetone_3 (format: TIFF)

7.1.2 Dimensions

Width: 1850 px

Height: 1694 px

7.1.3 Resolution

100 µm/px

7.1.4 Bit-depth

8-bit (256 colors)

7.1.5 Image location

TCA-acetone_3.tif

7.1.6 Standard image orientation

Yes

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