Name: GH vs wt

Description: ph 4- 7
DIGE

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2010-09-29

1.1.2 Responsible person or role

Affiliation: University of Munich

(i) Name: Andreas Blutke

(ii) Postal address: Institute of Veterinary Pathology
Ludwig-Maximilians-Universitaet München
Veterinärstr.13
80539 München
Germany.

(iii) Email address: blutke@patho.vetmed.uni-muenchen.de

1.1.3 Electrophoresis type

Difference gel electrophoresis

2. Sample

2.1.1 Sample Name(s)

    1. Sample name: GH tg glomeruli
    2. Sample type: Test sample
    1. Sample name: wt glomeruli
    2. Sample type: Control sample

2.1.2 Loading buffer

  1. 2 M Thiourea (Sigma, Germany) 7 M Urea (Roth, Germany) 30 mM Tris (Roth, Germany) 4 % (w/v) CHAPS (3-[Cholamidopropyl]dimethylammonio)-1- propanesulfonate) (Sigma, Germany) Adjust to pH 8.5 with 25 % HCl (Merck, Germany)

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 Bio Sciences
Model: Immobiline™ DryStrips, pH 4-7, 18 cm
Model number: 17-1233-01
Batch number: ???

3.2.3 Physical dimensions

X: 178 mm
Y: 3 mm
Z: 0.5 mm

3.2.4 Physiochemical property range and distribution

linear pH 4 - 7

3.2.5 Acrylamide concentration

7 %

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: GH tg glomeruli
  • Volume of sample: 120 µg
  • Loading buffer: 2 M Thiourea (Sigma, Germany) 7 M Urea (Roth, Germany) 30 mM Tris (Roth, Germany) 4 % (w/v) CHAPS (3-[Cholamidopropyl]dimethylammonio)-1- propanesulfonate) (Sigma, Germany) Adjust to pH 8.5 with 25 % HCl (Merck, Germany)
  • Volume of loading buffer: 50 µL

Loading method: cup loading.

3.3 Protocol

3.3.1 Buffers

No buffer.

3.3.2 Electrophoresis conditions

Running temperature: 20 °C

Hold: 0.1 mA, 12 h

4. Inter-dimension Process

4.1 Protocol

4.1.1 Step name

reduction

4.1.2 Inter dimension buffer

6 M Urea (Roth, Germany)
30 % (v/v) Glycerol (Roth, Germany)
2 % (w/v) SDS (Serva, Germany)
50 mM TrisHCl pH 6.8

4.1.3 Additional reagents

1% DTE

4.1.4 Equipment

4.1.5 Protocol

Temperature: 20 °C.

Duration: 15 min.

Protocol:
Shake 15 min

4.1.1 Step name

alkylation

4.1.2 Inter dimension buffer

6 M Urea (Roth, Germany)
30 % (v/v) Glycerol (Roth, Germany)
2 % (w/v) SDS (Serva, Germany)
50 mM TrisHCl pH 6.8

4.1.3 Additional reagents

2.5 % iodacetamide
200 µl bromphenol blue

4.1.4 Equipment

4.1.5 Protocol

Temperature: 20 °C.

Duration: 15 min.

Protocol:
Shake 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:

Separation gel (12 % polyacrylamide) (2 gels)
26.8 ml ddH2O
20 ml 1.5 M TrisHCl pH 8.8 (Roth, Germany)
32 ml Acrylamide / bisacrylamide (37.5:1) (Serva, Germany)
800 μl SDS 10 % (Serva, Germany)
400 μl Ammonium persulfate 10 % (Merck, Germany)
40 μl N,N,N’,N’-Tetramethylethylendiamine (TEMED) (Roth, Germany)

Stacking gel (4 % polyacrylamide) (2 gels)
6.1 ml ddH2O
2.5 ml 0.5 M TrisHCl pH 6.8 (Roth, Germany)
1.3 ml Acrylamide / bisacrylamide (37.5:1) (Serva, Germany)
100 μl SDS 10 % (Serva, Germany)
50 μl Ammonium persulfate 10 % (Merck, Germany)
10 μl N,N,N’,N’-Tetramethylethylendiamine (TEMED) (Roth, Germany)

3.2.3 Physical dimensions

X: 18 cm
Y: 16 cm
Z: 0.1 cm

3.2.4 Physiochemical property range and distribution

linear apparent molecular mass 100 - 10 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: well loading.

3.3 Protocol

3.3.1 Buffers

25 mM Tris (Roth, Germany)
192 mM Glycine (Roth, Germany)
0.1 % (w/v) SDS (Serva, Germany)

3.3.2 Electrophoresis conditions

Running temperature: 9 °C

Hold: 30 mA, 30 min

Hold: 40 mA, 3 h

5. Detection

5.1 Direct detection

5.1.1 Name of direct detection_process

Fluorescent staining

5.1.2 Direct detection agents

DIGE labels

5.1.3 Additional reagents and buffers

No additional reagents or buffer

5.1.4 Equipment

Manufacturer: GE Healthcare Bio Sciences
Model: Ettan™ DIGE Imager
Model number: 63-0056-42

5.1.5 Direct detection protocol

Temperature: 20 °C.

Duration: 30 min.

Protocol:
Scanning

6. Image Acquisition

6.1 Acquisition Equipment

6.1.1 Type of equipment

fluorescent scanner

6.1.2 Name of equipment

Manufacturer: GE Healthcare Bio Sciences
Model: Ettan™ DIGE Imager
Model number: 63-0056-42

6.1.3 Software

Manufacturer: GE Healthcare Bio Sciences
Model: Ettan™ DIGE Imager
Model number: 63-0056-42

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

According to manufacturer

6.2.2 Reference to gel matrix

There is only one gel in this document.

7. Image

7.1.1 Image name (or id)

GH_4-7 (format: TIFF)

7.1.2 Dimensions

Width: 1416 px

Height: 1400 px

7.1.3 Resolution

300 ppi

7.1.4 Bit-depth

8-bit (256 colors)

7.1.5 Image location

GH_4-7.tif

7.1.6 Standard image orientation

Yes

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