Name: pH 6-9 map of bovine placenta protein

Description: Description: We analyzed the protein expression of three normal placentae obtained from the afterbirth of AI-derived Korean Native Cattle fetuses born 280 days after insemination

Version: MIAPE: Gel Electrophoresis 1.4

1. General features

1.1.1 Date Stamp

2009-08-11

1.1.2 Responsible person or role

Affiliation: Professor

(i) Name: Jin, Dong IL

(ii) Postal address: 79 Daehangno, yoseong-gu, Daejeon, 305-764, Chungnam National University, Korea

(iii) Email address: dijin@cnu.ac.kr

1.1.3 Electrophoresis type

Two-Dimensional electrophoresis

2. Sample

2.1.1 Sample Name(s)

    1. Sample name: Bovine placenta tissue at late pregnancy
    2. Sample type: Standard

2.1.2 Loading buffer

  1. 1% SDS, 1 mM PMSF, 1X protease inhibitor cocktail [complete; Roche], 100 mM Tris-HCl(pH 7.0)
  2. 0.3% SDS, 3% DTT, 1 mM PMSF, protease inhibitor, 500 mM Tris-HCl(pH8.0)

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: immobiline Dry strip pH4-7, pH6-9
Model number: 17-1233-01, 17-6001-01
Batch number: unknown

3.2.3 Physical dimensions

X:
Y:
Z:

3.2.4 Physiochemical property range and distribution

-

3.2.5 Acrylamide concentration

%

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker:
Ratio: :

3.2.7 Additional substances in gel

No additional substance

3.2.8 Gel lane

1

3.2.9 Sample application

Lane 1

  • Sample: Bovine placenta tissue at late pregnancy
  • Volume of sample: 1 mg
  • Loading buffer: 0.3% SDS, 3% DTT, 1 mM PMSF, protease inhibitor, 500 mM Tris-HCl(pH8.0)
  • Volume of loading buffer: 110 µL

Loading method: cup loading.

3.3 Protocol

3.3.1 Buffers

No buffer.

3.3.2 Electrophoresis conditions

Running temperature: 25 °C

Hold: 50 V, 1000 min

Hold: 100 V, 100 min

Hold: 300 V, 100 min

Hold: 600 V, 100 min

Hold: 1000 V, 100 min

Hold: 3000 V, 100 min

Hold: 5000 V, 100 min

Hold: 8000 V, 1000 min

4. Inter-dimension Process

4.1 Protocol

4.1.1 Step name

equilibration

4.1.2 Inter dimension buffer

After the first dimensional IEF, IPG gel strip were placed in an equilibration solution (6 M urea, 2% SDS, 50% v/v glycerol, 2.5% acrylamide, 1.875 M Tris-HCl, pH 8.8) containing 5 mM TBP for 20min with gentle shaking.

4.1.3 Additional reagents

No additional reagent.

4.1.4 Equipment

Manufacturer: GE Healthcare
Model: etten DALT 2-D system
Model number: unknown

4.1.5 Protocol

Temperature: 25 °C.

Duration: 24 h.

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

N/A
Denaturing

3.2.2 Gel manufacture

3.2.3 Physical dimensions

X:
Y:
Z:

3.2.4 Physiochemical property range and distribution

-

3.2.5 Acrylamide concentration

%

3.2.6 Acrylamide : Crosslinker ratio

Crosslinker:
Ratio: :

3.2.7 Additional substances in gel

No additional substance

3.2.8 Gel lane

3.2.9 Sample application

Loading method: N/A.

3.3 Protocol

3.3.1 Buffers

*. 5X SDS Running Buffer (20L)
Glycine (amresco, 0167) 1440g
SDS (Lauryl Sulfate, Sigma, L-3771) 100g
Tris base (amresco, 0826) 300g
Water up to 20L

3.3.2 Electrophoresis conditions

Running temperature: 25 °C

Hold: 30 V, 3 h

Hold: 70 V, 20 h

5. Detection

5.1 Direct detection

5.1.1 Name of direct detection_process

Coomassie blue staining

5.1.2 Direct detection agents

colloidal Coomassie brilliant blue (CBB) G-250

5.1.3 Additional reagents and buffers

Fixer (2L)
MeOH (TEDIA, MS-1922) 800ml (net 40%)
Phosphoric Acid 100ml (net 5%)
Water 1100ml


Coomassie (1L)
Ammonium sulfate 170g (net 17%)
Phosphoric Acid 36ml (net 3%)
Coomassie G-250 (Fluka, 27815) 1g
MeOH (TEDIA, MS-1922) 340ml (net 34%)
Water up to 1L

5.1.4 Equipment

Manufacturer: unknown
Model: unknown
Model number: unknown

5.1.5 Direct detection protocol

Temperature: 25 °C.

Duration: 2 d.

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 1 min.
2. Soaked in fixer for 1 hour.
3. Soaked in CBB buffer for overnight.
4. Washed 3 times in deionized water.
5. Destained overnight in deionized water

Detection is described in the following reference protocol:
Citation: Proteomics,5, page(s) 4264-4273 (2005).
URL: not provided.

6. Image Acquisition

6.1 Acquisition Equipment

6.1.1 Type of equipment

GS-710 calibrated densitometer scanner

6.1.2 Name of equipment

Manufacturer: Bio-rad
Model: GS-710 calibrated densitometer
Model number: unknown

6.1.3 Software

Manufacturer: Swiss Institute for Bioinformatics, Geneva, Switzerland
Model: Melanie III
Model number: unknown

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

The stained gels were scanned at an optical resolution of 63.5 µm/pixel

6.2.2 Reference to gel matrix

There is only one gel in this document.

7. Image

7.1.1 Image name (or id)

pH6-9 (format: TIFF)

7.1.2 Dimensions

Width: 2340 px

Height: 2397 px

7.1.3 Resolution

215.254 ppi

7.1.4 Bit-depth

16-bit (HighColor)

7.1.5 Image location

Bovine placenta pH6-9(IDS).tif

7.1.6 Standard image orientation

Yes

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