Gel: Escherichia coli, pH 5-6 (Analytical 2-DE) (html display)

Name: Escherichia coli, pH 5-6 (Analytical 2-DE)

Description: Escherichia coli, pH 5-6 -Analytical 2-DE
Reference: L. Tonella, C. Hoogland, P.-A. Binz, R.D. Appel, D. F. Hochstrasser., J.-C. Sanchez. New perspectives in the Escherichia coli proteome investigation. Proteomics 2001, 1, 409-423. (doi:10.1002/1615-9861(200103)1:3<409::AID-PROT409>3.0.CO;2-M)

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2000-08-01

1.1.2 Responsible person or role

Affiliation: Swiss Institute of Bioinformatics

(i) Name: Christine Hoogland

(ii) Postal address: Swiss Institute of Bioinformatics CMU - 1, rue Michel Servet 1211 Geneva 4

(iii) Email address: christine.hoogland@isb-sib.ch

1.1.3 Electrophoresis type

Two-Dimensional electrophoresis

2. Sample

2.1.1 Sample Name(s)

1. Sample name: E. coli K12, strain W3110
2. Sample type: Test sample
3. URL: http://www.expasy.org/ch2d/protocols/protocols.fm1.html

2.1.2 Loading buffer

urea (8 M), CHAPS (4% w/v), Tris (40 mM), DTE (65 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: Pharmacia-Hoeffer
Model: IPGs NL pH 5-6
Model number: 17-6001-86
Batch number: unknown

3.2.3 Physical dimensions

X: 180 mm
Y: 3 mm
Z: 0.5 mm

3.2.4 Physiochemical property range and distribution

non-linear pH 5 - 6

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

3.2.9 Sample application

Lane 1

Sample: E. coli K12, strain W3110
Volume of sample: 100 µg
Loading buffer: urea (8 M), CHAPS (4% w/v), Tris (40 mM), DTE (65 mM) and a trace of bromophenol blue
Volume of loading buffer: 450 µL
Volume of mixture loaded in the lane: 450 µL

Loading method: well loading.
Additional comment: When the rehydration cassette had been thoroughly emptied and opened, the strips were transferred to the Pharmacia strip tray. After placing 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 [6].

3.3 Protocol

3.3.1 Buffers

No buffer.

3.3.2 Electrophoresis conditions

Running temperature: room temperature (no cooling device).

Gradient: 300-3500 V, 3 h

Hold: 3500 V, 3 h

Hold: 5000 V, 18 h

4. Inter-dimension Process

4.1 Protocol

4.1.1 Step name

equilibration

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-Hoeffer
Model: Immobiline strip tray
Model number: unknown

4.1.5 Protocol

Duration: 12 min.

Protocol:
100ml of this solution for 12min

4.1.1 Step name

SH_blocking

4.1.2 Inter dimension buffer

Tris-HCl (50 mM) pH 6.8, urea (6 M), glycerol (30% v/v), SDS (2% w/v), iodoacetamide (2.5% w/v) and a trace of Bromophenol Blue

4.1.3 Additional reagents

No additional reagent.

4.1.4 Equipment

Manufacturer: Pharmacia-Hoeffer
Model: Immobiline strip tray
Model number: unknown

4.1.5 Protocol

Duration: 5 min.

Protocol:
100 ml of solution 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: not provided.
URL: http://www.expasy.org/ch2d/protocols/protocols.fm3.html

3.2.3 Physical dimensions

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

3.2.4 Physiochemical property range and distribution

logarithmic apparent molecular mass 10 - 200 kDa

3.2.5 Acrylamide concentration

9-16 % (sigmoidal)

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: PDA
Ratio: 37.5:1

3.2.7 Additional substances in gel

Sodium thiosulfate 5mM
TEMED 0.05%
APS 0.1%

3.2.8 Gel lane

3.2.9 Sample application

Loading method: IPG_transfer.
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 70o 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, 5 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

Manufacturer: unknown
Model: orbital shaker
Model number: unknown

5.1.5 Direct detection protocol

Duration: 5 h.

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.

Detection is described in the following reference protocol:
Citation: not provided.
URL: http://www.expasy.org/ch2d/protocols/protocols.fm4.html

6. Image Acquisition

6.1 Acquisition Equipment

6.1.1 Type of equipment

laser scanner

6.1.2 Name of equipment

Manufacturer: Molecular Dynamics, CA
Model: Laser Densitometer (4000 x 5000 pixels; 12 bits/pixel)
Model number: unknown

6.1.3 Software

Manufacturer: Molecular Dynamics, CA
Model: ImageQuant
Model number: 3.0

6.1.4 Calibration

Yes (automatic)

6.1.5 Equipment specific parameters

unknown

6.2 Acquisition Protocol

6.2.1 Image acquisition process

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)

ECOLI5-6 (format: TIFF)

7.1.2 Dimensions

Width: 1011 px

Height: 1049 px

7.1.3 Resolution

300 dpi

7.1.4 Bit-depth

16-bit (HighColor)

7.1.5 Image location

ftp://ftp.expasy.org/databases/swiss-2dpage/MASTERS/ECOLI5-6.mel

7.1.6 Standard image orientation

Yes