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Name: Proteome of A549 cells

Description: 2D-gel of A549 cells (untreated), ph 4-9

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2007-05-01

1.1.2 Responsible person or role

Affiliation: UFZ-Helmholtz-Centre for Environmental Research

(i) Name: PD Dr. Martin von Bergen

(ii) Postal address: Department of Proteomics
Permoserstr. 15
04318 Leipzig
Germany

(iii) Email address: martin.vonbergen@ufz.de

1.1.3 Electrophoresis type

Two-Dimensional electrophoresis

2. Sample

2.1.1 Sample Name(s)

    1. Sample name: A549 cell lysate
    2. Sample type: Control sample

2.1.2 Loading buffer

  1. DeStreak Solution (GE Healthcare) with 0.5 % IPG buffer 3-10 NL

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 manufactured using the following reference protocol:

Citation: Proteomics,9, page(s) 4920–4933 (2009).
URL: not provided.

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 3 - 10

3.2.5 Acrylamide concentration

4 %

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 4:3

3.2.7 Additional substances in gel

No additional substance

3.2.8 Gel lane

1

3.2.9 Sample application

Lane 1

  • Sample: A549 cell lysate
  • Volume of sample: 500 µg
  • Loading buffer: DeStreak Solution (GE Healthcare) with 0.5 % IPG buffer 3-10 NL
  • Volume of loading buffer: 400 µL

Loading method: rehydration loading.

3.3 Protocol

3.3.1 Buffers

No buffer.

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Hold: 300 V, 6 h

Gradient: 300-1000 V, 6 h

Gradient: 1000-8000 V, 3 h

Hold: 8000 V, 9 h

4. Inter-dimension Process

4.1 Protocol

4.1.1 Step name

reduction

4.1.2 Inter dimension buffer

containing 6M urea, 30% glycerol, 4% SDS, 0.05M
Tris/HCl, bromophenol blue

4.1.3 Additional reagents

dte (20 mg/ml)

4.1.4 Equipment

4.1.5 Protocol

Temperature: 20 °C.

Duration: 15 min.

4.1.1 Step name

alkylation

4.1.2 Inter dimension buffer

containing 6M urea, 30% glycerol, 4% SDS, 0.05M
Tris/HCl, bromophenol blue

4.1.3 Additional reagents

iodoacetamide (25 mg/ml)

4.1.4 Equipment

4.1.5 Protocol

Temperature: 20 °C.

Duration: 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: Proteomics,9, page(s) 4920–4933 (2009).
URL: not provided.

3.2.3 Physical dimensions

X: 160 mm
Y: 200 mm
Z: 1 mm

3.2.4 Physiochemical property range and distribution

non linear apparent molecular mass 15 - 200 kDa

3.2.5 Acrylamide concentration

12 %

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker: Bisacrylamide
Ratio: 30:1

3.2.7 Additional substances in gel

No additional substance

3.2.8 Gel lane

1

3.2.9 Sample application

Loading method: the strip was placed on the gel and fixed with agarose .

3.3 Protocol

3.3.1 Buffers

0.3% (w/V) TRIS
1.44 % (w/V) Glycine
0.1% (w/V) SDS

3.3.2 Electrophoresis conditions

Running temperature: 12 °C

Hold: 12 mA, 30 min

Hold: 6 mA, 16 h

5. Detection

5.1 Direct detection

5.1.1 Name of direct detection_process

colloidal coomassie

5.1.2 Direct detection agents

Coomassie G250

5.1.3 Additional reagents and buffers

No additional reagents or buffer

5.1.4 Equipment

No specialised equipment.

5.1.5 Direct detection protocol

Detection is described in the following reference protocol:
Citation: electrophoresis,9, page(s) 255-62 (1988).
URL: not provided.

6. Image Acquisition

6.1 Acquisition Equipment

6.1.1 Type of equipment

flatbed scanner

6.1.2 Name of equipment

Manufacturer: GE Healthcare
Model: Image scanner II
Model number: 18-1170-84

6.1.3 Software

Manufacturer: Umax
Model: MagicScan
Model number: 4.6

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

-default scanner settings

6.2.2 Reference to gel matrix

There is only one gel in this document.

7. Image

7.1.1 Image name (or id)

a549 big with IDs.png (format: PNG)

7.1.2 Dimensions

Width: 1063 px

Height: 1094 px

7.1.3 Resolution

150 dpi

7.1.4 Bit-depth

32-bit (TrueColor)

7.1.5 Image location

a549 big with IDs.png

7.1.6 Standard image orientation

Yes

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