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Name: Test gel

Description: This is the protocol example for a 2-DE gel

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2004-09-04

1.1.2 Responsible person or role

Affiliation: SIB

(i) Name: Xavier Robin

(ii) Postal address: 1, rue Michel Servet, CH-1211 Genève 4, Switzerland

(iii) Email address: robin0@etu.unige.ch

1.1.3 Electrophoresis type

Two-Dimensional electrophoresis

2. Sample

2.1.1 Sample Name(s)

    1. Sample name: Colon epithelial cell (CEC)
    2. Sample type: Test sample
    3. URL: http://www.expasy.org/ch2d/protocols/protocols.fm1.html#998547

2.1.2 Loading buffer

  1. Urea (8 M), CHAPS (4 % w/v), DTE (65 mM), Tris (40 mM) 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: GE Healthcare
Model: IPGs NL pH 3.5-10
Model number: 17-1235-01
Batch number: unknown

3.2.3 Physical dimensions

X: 180 mm
Y: 3 mm
Z: 1.5 mm

3.2.4 Physiochemical property range and distribution

linear pH 3.5 - 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: Colon epithelial cell (CEC)
  • Volume of sample: 106 µg
  • Loading buffer: Urea (8 M), CHAPS (4 % w/v), DTE (65 mM), Tris (40 mM) and a trace of bromophenol blue.
  • Volume of loading buffer: 100 µL
  • Volume of mixture loaded in the lane: 100 µL

Loading method: cup loading.
Additional comment: Rehydration : Hydration was performed overnight in the Pharmacia reswelling cassette with 25 ml of a solution containing urea (8 M), CHAPS (2% w/v), DTE (10 mM), Resolyte pH 3.5-10 (2% v/v) and a trace of Bromophenol Blue.

After placing rehydrated IPG strips, humid electrode wicks, electrodes and sample cups in position, the strips and cups were covered with low viscosity paraffin oil. Samples were applied at the cathodic end of the IPG strips in a slow and continuous manner, without touching the gels.

3.3 Protocol

3.3.1 Buffers

No buffer.

3.3.2 Electrophoresis conditions

Running temperature: 8-12 °C

Gradient: 300-3500 V, 3 h

Hold: 3500 V, 3 h

Hold: 5000 V, 8 h

4. Inter-dimension Process

4.1 Protocol

4.1.1 Step name

equilibration (resolubilization and reduction)

4.1.2 Inter dimension buffer

Tris-HCl (50mM) pH 8.4.

4.1.3 Additional reagents

Urea (6 M), glycerol (30% v/v), SDS (2% w/v) and DTE (2% w/v)

4.1.4 Equipment

Manufacturer: Pharmacia
Model: Strip tray
Model number: unknown

4.1.5 Protocol

Duration: 12 min.

Protocol:
The strips were equilibrated within the strip tray with 100 ml of the solution described above (4.1.2, 4.1.3) for 12 min.

4.1.1 Step name

blocking

4.1.2 Inter dimension buffer

Tris-HCl (50mM) pH 6.8.

4.1.3 Additional reagents

Urea (6 M), glycerol (30% v/v), SDS (2% w/v), iodoacetamide (2.5% w/v) and a trace of Bromophenol blue.

4.1.4 Equipment

Manufacturer: Pharmacia
Model: Strip tray
Model number: unknown

4.1.5 Protocol

Duration: 5 min.

Protocol:
-SH groups were subsequently blocked with 100 ml of the solution described above (4.1.2, 4.1.3) for 5 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: Anal. Biochem.,173, page(s) 412-423 (1988).
URL: not provided.

3.2.3 Physical dimensions

X: 160 mm
Y: 200 mm
Z: 1.5 mm

3.2.4 Physiochemical property range and distribution

linear apparent molecular mass 20 - 200 kDa

3.2.5 Acrylamide concentration

9 - 16 % (linear)

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: PDA
Ratio: 37.5:1

3.2.7 Additional substances in gel

TEMED 0.05%
Sodium thiosulfate 5mM
APS 0.1%

3.2.8 Gel lane

1

3.2.9 Sample application

Loading method: gel loading.
Additional comment: After the equilibration, the IPG gel strips were cut to size. Six mm were removed from the anodic end and 14 mm from the cathodic end. The second dimension gels were overlayered with a solution containing agarose (0.5% w/v) and Tris-glycine-SDS (25 mM-198 mM-0.1% w/v) pH 8.3 heated at about 70°C and the IPG gel strips were immediately loaded through it.

3.3 Protocol

3.3.1 Buffers

Running buffer: Tris-Glycine-SDS (25 mM - 198 mM - 0.1% w/v) pH 8.3
Leading buffer: Tris-HCl (0.375 M) pH 8.8.

3.3.2 Electrophoresis conditions

Running temperature: 8-12 °C

Hold: 40 mA, 4 h

5. Detection

5.1 Direct detection

5.1.1 Name of direct detection_process

Ammoniacal silver staining

5.1.2 Direct detection agents

6g silver

5.1.3 Additional reagents and buffers

Solution 1: ethanol: acetic acid: water (40: 10: 50)
Solution 2: ethanol: acetic acid: water (5: 5: 90)
Solution 3: glutaraldehyde (1%) and sodium acetate (0.5 M)
Solution 4: 2,7 naphtalene-disulfonic acid solution (0.05% w/v)
Solution 5: 6 g of silver nitrate were dissolved in 30 ml of deionized water, which was slowly mixed into a solution containing 160 ml of water, 10 ml of concentrated ammonia (25%) and 1.5 ml of sodium hydroxide (10 N). A transient brown precipitate might form. After it cleared, water was added to give the final volume.
Solution 6: citric acid (0.01% w/v) and formaldehyde (0.1% v/v)
Solution 7: Tris (5% w/v) and acetic acid (2% v/v)

5.1.4 Equipment

No specialised equipment.

5.1.5 Direct detection protocol

Protocol:
1. At the end of the second dimension run, the gels were removed from the glass plates and washed in deionized water for 5 min.
2. Soaked in Solution 1 for 1 hour.
3. Soaked in Solution 2 for 2 hours or overnight.
4. Washed in deionized water for 5 min.
5. Soaked in Solution 3 for 30 min.
6. Washed 3 times in deionized water for 10 min.
7. In order to obtain homogeneous dark brown staining of proteins, gels were soaked twice in 750 ml Solution 4 for 30 min.
8. Rinsed 4 times in deionized water for 15 min.
9. Gels were stained in a freshly made Solution 5 for 30 minutes.
10. After staining, the gels were washed 4 times in deionized water for 4 min.
11. The images were developed in Solution 6 for 5 to 10 min.
12. When a slight background stain appeared, development was stopped with Solution 7.

6. Image Acquisition

6.1 Acquisition Equipment

6.1.1 Type of equipment

camera

6.1.2 Name of equipment

Manufacturer: Molecular Dynamics
Model: Laser Densitometer
Model number: unknown

6.1.3 Software

Manufacturer: GE Healthcare
Model: ImageQuant
Model number: unknown

6.1.4 Calibration

Yes (automatic)

6.1.5 Equipment specific parameters

Parameters used are now unknown

Configuration file: Configuration file for the software

6.2 Acquisition Protocol

6.2.1 Image acquisition process

Protocol used is now unknown

6.2.2 Reference to gel matrix

There is only one gel in this document.

7. Image

7.1.1 Image name (or id)

CEC_HUMAN.mel (format: TIFF)

7.1.2 Dimensions

Width: 5000 px

Height: 4000 px

7.1.3 Resolution

20 px/mm

7.1.4 Bit-depth

12-bit

7.1.5 Image location

CEC_HUMAN.mel

7.1.6 Standard image orientation

Yes

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